Microfluidic Products with Controlled Fluid Flow

ABSTRACT

A microfluidic product utilizing gradient surface energy coatings for fluid control comprising a plurality of fluid passages wherein at least one fluid passage comprises a coating configured to control liquid flow wherein the coating configured to control liquid flow comprises a gradient surface energy coating from a proximal location to a distal location on a surface of the fluid passage. The product can include uniform regions and surface gradient regions in the same passage. Coating compositions and product dimensions can be selected to provide control over different flow properties including fluid velocity, reduction and acceleration of fluid flow, and starting and stopping fluid flow.

RELATED APPLICATIONS

This application is a continuation application that claims benefit ofU.S. patent application Ser. No. 15/973,882 filed May 8, 2018 whichclaims the benefit of U.S. patent application Ser. No. 14/31,889 filedSep. 25, 2015 (now U.S. Pat. No. 9,968,930), a 371 of PCT/US2014/031889filed Mar. 26, 2014 that claims priority from provisional application61/808,292 filed Apr. 4, 2013 and provisional application 61/859,652filed Jul. 29, 2013 the contents of which are herein incorporated byreference in their entirety.

FIELD OF INVENTION

The present invention relates to microfluidic products using surfaceenergy gradients to control fluid flow within the product.

BACKGROUND

The use of microfluidic technology is suitable for a number ofanalytical chemical and biochemical operations. These technologiesprovide advantages of being able to perform chemical and biochemicalreactions, macromolecular separations, and the like, that range from thesimple to the relatively complex, in automatable, high-throughput,low-volume systems. In particular, these systems employ networks ofintegrated microscale channels in which materials are transported,mixed, separated and detected. The small size of these systems allowsfor the performance reactions at substantially greater rates, and withsubstantially less reagent volume. Expensive or rare fluids are employedin many emerging scientific applications, such as proteomics andgenomics. Thus, considerable interest has been focused on microfluidictechniques, which typically involve small sample volumes and low reagentconsumption. In addition, microfluidic techniques may be used to carryout numerous parallel processes, can be used across a range of fluidproperties, and are compatible with movement of biological moieties thatmay vary by orders of magnitude in size and physical characteristics(e.g., from peptide hormones to intact cells).

A variety of microfluidic devices have been developed for chemical andbioanalytical applications. In some applications, microfluidic devicesinvolve the miniaturization and automation of a number of laboratoryprocesses, which are then integrated on a chip. Thus, microfluidictechnology may be employed to carry out a series of chemical orbiochemical processes in a single device, including sample purification,separation, and detection of specific analytes. Applications includemedical diagnostics, genetic analysis, or environmental sampling. See,e.g., Ramsey et al. (1995) “Microfabricated chemical measurementsystems,” Nat. Med. 1:1093-1096.

Typical microfluidic systems employ a body structure or substrate thathas at least one microscale channel disposed within it. Examples of suchsystems range from simple tubular capillary systems, e.g., fused silicacapillaries, to more complex planar devices that can have from one toseveral intersecting channels disposed therein, i.e., between at leasttwo planar substrate layers. Microfluidic systems generally have a broadrange of uses including separation and characterization ofmacromolecular species, e.g., proteins and nucleic acids, see e.g., U.S.Pat. No. 5,699,157, screening assay platforms, e.g., drug screening,diagnostics, etc. Substrates and/or cover plates can be comprised of arigid material such as glass (see, e.g., Woolley et al. (1994),“Ultra-high-speed DNA fragment separations using microfabricatedcapillary array electrophoresis chips,” Proc. Natl. Acad. Sci. USA91:11348-11352), plastic (see, e.g., McCormick et al. (1997),“Microchannel electrophoretic separations of DNA in injection-moldedplastic substrates,” Anal. Chem. 69:2626-2630), silicon, or quartz. Inother applications, microfabricated elastomeric valve and pump systemshave been proposed in International Patent Publication No. WO01/01025.Similar valves and pumps are also described in Unger et al. (2000)“Monolithic microfabricated valves and pumps by multilayer softlithography,” Science 288:113-116. These publications describe softlithography as an alternative to silicon-based micromachining as a meansby which to form microfluidic devices.

The above-described microfluidic devices, however, pose certaintechnical challenges that must be overcome. For example, fluid flowcharacteristics within the small flow channels of a microfluidic devicemay differ from the flow characteristics of fluids in larger devices,since surface effects tend to predominate, and regions of bulk flowbecome proportionately smaller. Several techniques have been developedin order to achieve fluid flow control in microfluidic devices. Onetechnique involves the generation of electric fields to manipulatebuffered, conductive fluids around networks of channels throughelectrophoretic or electroosmotic forces. See, e.g., Culbertson et al.(2000), “Electroosmotically induced hydraulic pumping on microchips:differential ion transport,” Anal. Chem. 72:2285-2291. Anothertechnique, as described in Anderson et al. (2000), “A miniatureintegrated device for automated multistep genetic assays,” Nucleic AcidsRes. 28:E60, describes fluidic control by coupling the device to anexternal system of solenoid valves and pressure sources. However, thesefluid control mechanisms greatly increase the complexity, cost, andmanufacturability of such highly integrated designs.

Typically, microfluidic devices employ fluid or material directionsystems to transport fluids or other materials through and among thechannels and chambers of the device in order to perform thecombinations, separations or other operations in carrying out a givenanalysis. Examples of such transport systems include pneumatically orhydraulically driven systems, e.g., as described in published PCTApplication No. 97/02357, systems incorporating microfabricated pumpsand/or valves, and, in preferred aspects, electrokinetic materialtransport systems, e.g., as described in Published PCT Application No.96/04547.

Wetting behavior of a liquid on a substrate surface is typically afunction of the surface energy of the substrate surface and the surfacetension of the liquid. At the liquid-substrate surface interface, if themolecules of the liquid have a stronger attraction to the molecules ofthe substrate surface than to each other (the adhesive forces arestronger than the cohesive forces), then wetting of the substratesurface generally occurs. Alternatively, if the molecules of the liquidare more strongly attracted to each other than to the molecules of thesubstrate surface (the cohesive forces are stronger than the adhesiveforces), then the liquid generally beads-up and does not wet the surfaceof the substrate. One way to quantify surface wetting characteristics ofa liquid on a surface of a substrate is to measure the contact angle ofa drop of liquid placed on that surface. The contact angle is the angleformed by the solid/liquid interface and the liquid/vapor interfacemeasured from the side of the liquid. Typically, a decrease in thecontact angle between the liquid and the surface correlates with anincrease in wetting.

For many applications (e.g., sensors and microfluidic devices), theability to precisely control the wetting and/or flow of a liquid on asurface of a substrate according to a precise high-resolution patterncan be important. Thus, it would be desirable to have additional methodsand materials that can provide such control.

Surface energy gradients are useful for transporting small fluid volumesin analytical or medical devices while reducing or eliminating externalforces. A microfluidic product using these gradients needs less energyto operate and could be shrunk to smaller sizes to be less invasive. Inaddition, the use of surface energy gradients to control fluid flow,including stopping and initiating flow within the microfluidic productcan reduce or eliminate the need for expensive pumps and controllers inthe overall system, greatly reducing the cost of current systems. Amicrofluidic product utilizing these gradients could be produced atsimilar or lower cost than current products and would also reduce thecost and complexity of external hardware and also the size of anyindividual components (analytical slides, cartridges, etc.). Inaddition, because the gradients can be created with small, precisedimensions, a component utilizing one or more surface energy gradientscan also reduce the amount of solution used in the system. Because ofthe improved fluid transport properties due the surface energygradients, the amount of solution loss due to hold-up in channels,wells, passages, etc. would also be greatly reduced.

New devices using surface energy gradients would have a great benefit.The invention has particular value for product applications that usehigh-volume, disposable parts.

SUMMARY

An embodiment of the invention is a microfluidic product that usessurface energy gradients to control fluid flow within channels and otherfluid passages (tubes, shunts, other cross-section geometries, etc). Thecomposition of the gradients as well as the degree of the gradient canbe adjusted to control different aspects of fluid flow, including fluidvelocity and stopping and starting fluid flow. The microfluidic productcan use one or more gradient compositions in a plurality of channels toprovide for different flow rates within different channels on a singleproduct.

One embodiment is a microfluidic product that utilizes surface energygradients for fluid control comprising a plurality of fluid passageswherein the fluid passages each comprise a top and a bottom surfacewherein at least one fluid passage comprises a gradient surface energyregion beginning at a proximal location on a surface of the fluidpassage and ending at a distal location on a surface of the fluidpassage. The product can include uniform regions and surface gradientregions in the same passage. Coating compositions and product dimensionscan be selected to provide control over different flow propertiesincluding fluid velocity, reduction and acceleration of fluid flow, andstarting and stopping fluid flow.

In an embodiment, the microfluidic product comprises one or more fluidpassages wherein a first fluid passage comprises a top and a bottomsurface wherein the first fluid passage comprises a coating configuredto control liquid flow wherein the coating comprises a gradient surfaceenergy coating from a proximal location to a distal location on asurface of the fluid passage. In an embodiment, the microfluidic productcomprises a plurality of fluid passages wherein the plurality of fluidpassages comprise a first fluid passage and a second fluid passage, eachwith a top and a bottom surface, wherein both the first fluid passageand the second fluid passage comprise a coating configured to controlliquid flow wherein the coating comprises a gradient surface energycoating from a proximal location to a distal location on a surface ofthe fluid passage. In an embodiment, the contact angle formed with waterand a surface at a proximal location of the surface energy gradient inthe first fluid passage is different from the contact angle formed withwater and a surface at a proximal location of the surface energygradient in the second fluid passage. In an embodiment, the contactangle formed with water and a surface at a distal location of thesurface energy gradient in the first fluid passage is different from thecontact angle formed with water and a surface at a distal location ofthe surface energy gradient in the second fluid passage. In anembodiment, the first fluid passage and the second fluid passage are influid communication with each other. In an embodiment, the microfluidicproduct further comprises a fluid passage that is not coated. Inembodiments, the fluid passages comprise rectangular or non-rectangularchannels. In embodiments, the fluid passages comprise circular channels.In embodiments, the fluid passages comprise non-circular channels. Inembodiments, the coating configured to control liquid flow is on thebottom surface of the fluid passage. In embodiments, the coatingconfigured to control liquid flow is on the top surface of the fluidpassages. In embodiments, the top and bottom surfaces of the fluidpassages are coated with different coating compositions.

U.S. Pat. No. 7,790,265 discloses surface energy gradients comprised ofmixed monolayer films and discloses different methods for producing suchgradients. The entire content of U.S. Pat. No. 7,790,265 patent isincorporated by reference into this application.

The surface can be a wide variety of materials including metals,glasses, plastics, ceramics, etc. In addition, the surface can be a basesubstrate of either rigid or flexible material that contains a basecoating. The base coating can be metallic, ceramic, or polymeric.

In an embodiment the surface is a nonwoven material or a film or otherflexible material. In an embodiment, the nonwoven material or filmcomprises a metallic coating such as aluminum, nickel, gold, silver,copper, or other materials. Multiple methods can be used to applymetallic coatings to different film or nonwoven surfaces.

One embodiment of the invention is an analytical device wherein thesurface energy gradient resides on a flexible film and the film isattached to or placed in contact with the top or bottom surface of aplastic material containing channels and/or wells intended for fluidtransport and/or analysis. The film can be used to seal the top orbottom of the channel (or both the top and bottom) to form a device withchannels containing a surface energy gradient on at least one surface.The width of the gradient coating can be different from the width of thechannel. In some instances, it may be desirable to keep the dropconfined to the gradient region without touching the plastic,non-gradient walls of the channel. In other instances, it may bedesirable from a manufacturing standpoint to manufacture the surfaceenergy gradients in the film with a wider width than the width of thechannels and overlay and seal the film over the channels.

There are multiple embodiments of devices that can utilize these methodsand designs. Examples of such devices include those disclosed in USpatent application US 2008/087868, “Method and Device for Forming anAssembly” filed Dec. 20, 2008 which is herein incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the initial contact angle required to initiateflow with water in channels with channel widths ranging from 200 micronto 2000 microns (2 mm).

FIG. 2A is a figure showing a microfluidic product used to test flowthrough channels.

FIG. 2B is a figure showing the layers used to make the product shown inFIG. 2A.

FIG. 3 is a graph showing how the fluid velocity was controlled usingdifferent surface energy gradient compositions

FIG. 4 is a graph showing further control of fluid flow within thechannels using surface energy gradient coatings.

FIG. 5 is a figure showing one embodiment of an inlet port for use inthe microfluidic products of the invention

FIG. 6 is a figure showing an embodiment of a pattern on a glass slide

FIG. 7 is a figure of one embodiment of a microfluidic product of theinvention

DETAILED DESCRIPTION

Current diagnostic and other systems using microfluidic cartridges areplagued by high hardware cost and/or poor control over fluid flow. Highhardware costs are often primarily due to expensive pumps and controlsystems which can be 80 percent or more of the system cost. Flow incartridges is typically disrupted by bubbles and other surfacevariances; and relatively long (sometimes serpentine) fluid pathsincrease the difficulty in controlling fluid flows. In addition, longfluid path lengths result in higher use of fluid and higher waste offluid due to hold-up within the channels and/or passages.

There is a need to control the delivery, flow control, and analysis ofprecise volumes of fluid within microfluidic devices. Surface energygradients can be used to improve or provide these functions within amicrofluidic device. The microfluidic products of the invention canimprove the accuracy and performance of diagnostic and other systemswhile also reducing their size. In an embodiment, separate channels inthe microfluidic product are coated with different gradient compositionsto provide for different fluid flow rates in the separate channels. Inan embodiment, at least one fluid passage comprises a coating configuredto control liquid flow wherein fluid flow is initiated at the beginningof the passage, and fluid flow velocity is stopped or reduced by atleast 50 percent within the passage. In an embodiment, at least onefluid passage comprises a coating configured to control liquid flowwherein fluid flow is initiated at the beginning of the passage, andfluid flow velocity is increased by at least 10 percent within thepassage. In an embodiment, at least one fluid passage comprises acoating configured to control liquid flow wherein fluid flow isinitiated at the beginning of the passage, and fluid flow velocity isincreased by at least 20 percent within the passage. In an embodiment,at least one fluid passage comprises a coating configured to controlliquid flow wherein fluid flow is initiated at the beginning of thepassage, and fluid flow velocity is increased by at least 100 percentwithin the passage. In an embodiment, the invention allows for fluids tobe moved through microfluidic products without using pumps or controlsystems. In an embodiment, at least one fluid passage comprises acoating configured to control liquid flow wherein fluid flow isinitiated at the beginning of the passage, and fluid flow velocity isdecreased by at least 50 percent within the passage. In an embodiment,at least one fluid passage comprises a coating configured to controlliquid flow wherein fluid flow is initiated at the beginning of thepassage, and fluid flow velocity is decreased by at least 10 percentwithin the passage. In an embodiment, at least one fluid passagecomprises a coating configured to control liquid flow wherein fluid flowis initiated at the beginning of the passage, and fluid flow velocity isdecreased by at least 20 percent within the passage. In an embodiment,at least one fluid passage comprises a coating configured to controlliquid flow wherein fluid flow is initiated at the beginning of thepassage, and fluid flow velocity is decreased by at least 100 percentwithin the passage. In an embodiment, the invention allows for fluids tobe moved through microfluidic products without using pumps or controlsystems. In an embodiment, a plurality of channels of the microfluidicproduct are in liquid communication with each other but the fluid flowvelocities in the channels are different.

Coatings can also be applied to the areas of the product that areadjacent to the entrance and exit of the channels. The composition ofthe coating in the areas adjacent to the entrances and exits of thechannels can be selected based on the composition of the coating insidethe channel and the fluid flow properties desired. In an embodiment, theareas of the microfluidic product that are adjacent to the entrance andexit of the channel are coated. In another embodiment, areas of themicrofluidic product that are adjacent to the entrance and exit of thechannel are not coated.

A wide range of methods can be used to create the coatings on themicrofluidic products. In self-assembled patterning the physicalchemical properties of a molecule or combination of molecules areexploited under specific conditions to produce distributions ofmolecules with known properties. For example, a self-assembled monolayerof alkanethiols on gold will often have a high degree of order thatresults from intermolecular interactions between the components of themolecules. In directed patterning, the position of molecules iscontrolled by information that is brought in from the outside, such as amask or a template. Directed patterning methods can in turn be dividedinto two types: lithographic approaches and writing approaches.Lithographic approaches include methods where a physical template suchas a mask or a mold is used to transfer a pattern to an object. Examplesinclude conventional photolithography and microcontact printing. Incontrast, writing approaches use a serial approach to transfer apattern, typically from a computer-based representation such as a CAD(computer assisted design) drawing, to an object. Electron beamlithography is a writing approach, by the definition used here. Ingeneral, lithographic approaches can produce many copies of the samepattern; writing approaches are often used for producing unique patternsfor producing a large number of different patterns, or for changingpatterns quickly. Useful digital application methods include, forexample, spray jet, valve jet, and inkjet printing methods. Techniquesand formulation guidelines are well known (see, for example,“Kirk-Othmer Encyclopedia of Chemical Technology”, Fourth Edition(1996), volume 20, John Wley and Sons, New York, pages 112-117, thedisclosure of which is incorporated herein by reference) and are withinthe capability of one of ordinary skill in the art. Combinations ofthese methods may also be employed.

Fluid materials used in practice of the present invention may be appliedto any portion of the substrate surface by various techniques including,for example, moving the substrate relative to a fixed applicator, or bymoving an applicator relative to the substrate. Accordingly, methods ofthe current invention are capable of forming detailed coating patternson a surface of a microfluidic product.

In an embodiment of the microfluidic product of the invention, the widthof the coating configured to control liquid flow is substantially equalto the width of the fluid passage. In some embodiments, the width of thecoating configured to control liquid flow can be greater or less thanthe width of the fluid passages. In an embodiment, the coatingconfigured to control liquid flow comprises a surface energy gradientregion. In some instances, it may be desirable to keep the liquidconfined to the surface energy gradient region without touching thewalls of the fluid passage. In other instances, it may be desirable (forexample, from a manufacturing standpoint) to manufacture the coatingswith a wider width than the width of the fluid passages.

A “microfluidic product” is defined as any product that comprises one ormore fluid passages wherein the volume capacity of any single fluidpassage is no more than than 100 microliters. In an embodiment, at leastone fluid passage has a volume of no more than 50 microliters. In anembodiment, at least one fluid passage has a volume of no more than 20microliters. In an embodiment, at least one fluid passage has a volumeof no more than 5 microliters. In an embodiment, at least one fluidpassage has a volume of no more than 1 microliter.

The capillary pressure within a channel can be predicted by thefollowing equation.

P=gam/h*(cos(thet1)+cos(thet2)−(2*h/w))  Eq 1)

where

-   -   P=Pressure    -   gam=surface tension of the liquid    -   h=channel height, distance between top and bottom surfaces    -   thet1=contact angle between liquid and bottom surface    -   thet2=contact angle between liquid and top surface    -   w=width of channel

To initiate flow into a channel, the capillary pressure must be greaterthan 0. As can be seen from the equation, capillary pressure is afunction of the contact angle the liquid forms with the top and bottomsurfaces, the channel dimensions, and the liquid surface energy. Each ofthese parameters can be adjusted to control the entrance of a liquidinto a capillary channel. Similar equations for different geometricshapes and varying cross-sections can be derived. A graph of capillarypressure as calculated in Equation 1 as a function of contact angle willshow that the relationship between capillary pressure and contact angleis approximately linear for contact angle values between 30 and 130degrees. This relationship indicates that velocity can be controlledwithin a channel by changing the contact angle in a controlled manner.Using the equations for drag in a capillary channel along with equation1 for calculating the capillary pressure, coating compositions can beconfigured to produce controlled surface energy gradient coatings thatcan be used to provide a controlled, or even constant, fluid velocityover the length of a channel. Coatings can be configured to controlliquid flow by providing coating compositions that control the initialcontact angle at the beginning of the channel, by providing coatingcompositions that change the contact angle over the length of thechannel, and by providing coating compositions that increase or decreasethe rate of change of the contact angle over the length of the channel.The degree of the surface energy gradient can be changed by changing thecoating composition over the length of the channel. For example, acoating could be configured on one channel surface that would provide acontact angle of 100 degrees at the entrance to a channel and that wouldthen reduce the contact angle by 10 degrees every 5 mm for a givenlength of the channel. To increase or decrease the velocity in differentchannels, coatings can be configured that change the rate of the contactangle change over the length of the channel—to provide a higher fluidvelocity, the rate of change in the contact angle per length could beincreased; to provide a lower fluid velocity, the rate of change in thecontact angle could be decreased. Average liquid velocity in the fluidchannels due to the change in capillary pressure provided by the surfaceenergy gradient can be varied from a low of 0 mm/s to more than 15 cm/sdepending on the fluid properties and the channel dimensions. Inembodiments, the average fluid velocity can be over 30 cm/s. The averagefluid velocity can also be increased further by increasing the liquidhead pressure from the fluid.

The microfluidic product can comprise one or more channels having a topand a bottom surface wherein the channel comprises a coating configuredto control liquid flow wherein the coating comprises a gradient surfaceenergy coating from a proximal location to a distal location on a top orbottom surface of the channel. In an embodiment, the microfluidicproduct can comprise a plurality of channels wherein the plurality ofchannels further comprise a first channel and a second channel whereinthe first channel and the second channel are in fluid communication witheach other. In an embodiment, the plurality of channels comprise a firstchannel and a second channel each with a top and a bottom surfacewherein the first channel and the second channel are in fluidcommunication with each other and wherein both the first channel and thesecond channel comprise a coating configured to control liquid flowwherein the coating comprises a gradient surface energy coating from aproximal location to a distal location on a surface of the channel. Inan embodiment, the composition of the coating in the first channel isdifferent from the composition of the coating in the second channel.

Surface energy gradients can be characterized by the contact angle thatwater forms with the coated surface; for surfaces comprising a surfaceenergy gradient region, the contact angle formed with water and thesurface at a proximal location of the region will be different than thecontact angle formed with water and the surface at a distal end of theregion. In embodiments, the difference between the contact angle and asurface at the proximal location of the region with the surface energygradient and the contact angle at a surface at the distal location ofthe region with the surface energy gradient will be at least 10 degrees.In embodiments, the difference between the contact angle and a surfaceat the proximal location of the region with the surface energy gradientand the contact angle at a surface at the distal location of the regionwith the surface energy gradient will be at least 20 degrees. Inembodiments, the difference between the contact angle and a surface atthe proximal location of the region with the surface energy gradient andthe contact angle at a surface at the distal location of the region withthe surface energy gradient will be at least 30 degrees. In embodiments,the difference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 40 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 50 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 60 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 70 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 80 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 90 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 100 degrees. In embodiments, thedifference between the contact angle and a surface at the proximallocation of the region with the surface energy gradient and the contactangle at a surface at the distal location of the region with the surfaceenergy gradient will be at least 110 degrees.

In an embodiment, the contact angle formed between water and a surfaceat the proximal location of the surface energy gradient region isgreater than 120 degrees. In an embodiment, the contact angle formedbetween water and a surface at the proximal location of the surfaceenergy gradient region is greater than 100 degrees. In an embodiment,the contact angle formed between water and a surface at the proximallocation of the surface energy gradient region is greater than 60degrees. In an embodiment, the contact angle formed between water and asurface at the proximal location of the surface energy gradient regionis greater than 40 degrees. In an embodiment, the contact angle formedbetween water and a surface at the proximal location of the surfaceenergy gradient region is greater than 20 degrees. In embodiments, thecontact angle formed between water and a surface at the proximallocation of the surface energy gradient region is between 90 and 120degrees and the contact angle formed between water and a surface at thedistal location of the region is between 10 and 110 degrees. Inembodiments, the contact angle formed between water and a surface at theproximal location of the region is between 90 and 120 degrees and thecontact angle formed between water and a surface at the distal locationof the region is between 60 and 110 degrees. In embodiments, the contactangle formed between water and a surface at the proximal location of theregion is between 90 and 120 degrees and the contact angle formedbetween water and a surface at the distal location of the region isbetween 10 and 70 degrees. In embodiments, the contact angle formedbetween water and a surface at the proximal location of the region isbetween 50 and 90 degrees and the contact angle formed between water anda surface at the distal location of the region is between 10 and 80degrees. In embodiments, the contact angle formed between water and asurface at the proximal location of the region is between 50 and 90degrees and the contact angle formed between water and a surface at thedistal location of the region is between 10 and 40 degrees. Inembodiments, the contact angle formed between water and a surface at theproximal location of the region is between 50 and 90 degrees and thecontact angle formed between water and a surface at the distal locationof the region is between 30 and 80 degrees. In embodiments, the contactangle formed between water and a surface at the proximal location of thesurface energy gradient region is between 40 and 100 degrees and thecontact angle formed between water and a surface at the distal locationof the region is between 10 and 100 degrees. In embodiments, the contactangle formed between water and a surface at the proximal location of theregion is between 40 and 100 degrees and the contact angle formedbetween water and a surface at the distal location of the region isbetween 10 and 70 degrees. In embodiments, the contact angle formedbetween water and a surface at the proximal location of the region isbetween 40 and 100 degrees and the contact angle formed between waterand a surface at the distal location of the region is between 10 and 40degrees.

Fluid flow can be decelerated or stopped by configuring the coating sothat the contact angle formed with water and a surface at a distallocation of the surface energy gradient region is higher than thecontact angle formed with water and a surface at a proximal end of thesurface gradient region. In embodiments, the contact angle formedbetween water and a surface at the proximal location of the surfaceenergy gradient region is at least 10 degrees lower than the contactangle formed between water and a surface at a distal location of theregion. In embodiments, the contact angle formed between water and asurface at the proximal location of the surface energy gradient regionis at least 30 degrees lower than the contact angle formed between waterand a surface at a distal location of the region. In embodiments, thecontact angle formed between water and a surface at the proximallocation of the surface energy gradient region is at least 50 degreeslower than the contact angle formed between water and a surface at adistal location of the region. In embodiments, the contact angle formedbetween water and a surface at the proximal location of the surfaceenergy gradient region is at least 90 degrees lower than the contactangle formed between water and a surface at a distal location of theregion.

In an embodiment, the microfluidic product comprises a plurality ofchannels wherein the plurality of channels comprise a first channel anda second channel each with a top and a bottom surface wherein both thefirst channel and the second channel comprise a coating configured tocontrol liquid flow wherein the coating comprises a gradient surfaceenergy coating from a proximal location to a distal location on asurface of the channel. In an embodiment, the contact angle formed withwater and a surface at a proximal location of the surface energygradient in the first channel is different from the contact angle formedwith water and a surface at a proximal location of the surface energygradient in the second channel. In an embodiment, the contact angleformed with water and a surface at a distal location of the surfaceenergy gradient in the first channel is different from the contact angleformed with water and a surface at a distal location of the surfaceenergy gradient in the second channel. In an embodiment, the firstchannel and the second channel are in fluid communication with eachother. In an embodiment, the microfluidic product further comprises achannel that is not coated.

Coating compositions can be configured to produce controlled surfaceenergy gradient coatings that can be used to provide a controlled, oreven constant, fluid velocity over the length of a channel. By providingdifferent individual channels with different coating compositions, thesurface energy gradient can be varied in each channel, resulting indifferent linear velocities for the fluid in individual channels. Thisfeature allows for a slow reaction or process to take place in a firstchannel and a faster reaction or process to take place in a secondchannel, all on the same layer of the microfluidic product while keepingthe same length, width, and height dimensions for the first and secondchannel. In some cases, serpentine paths and other long flow paths canbe eliminated from the design of the microfluidic product, resulting insimpler manufacturing designs and smaller overall product dimensions.Using coatings providing surface energy gradients, linear velocities forliquids such as water in the range of 0-30 cm/s can be achieved formicrofluidic channels. In an embodiment, linear velocity for the fluidin a first channel is greater than 0.5 mm/sec while linear velocity forthe fluid in a second channel is less than 0.1 mm/sec. In an embodiment,the microfluidic product comprises a plurality of channels comprising afirst channel and a second channel, each channel having a first surfaceand a second surface, wherein at least the first channel comprises acoating configured to control liquid flow from a proximal location to adistal location, and the linear velocity of fluid in the first channelis different from the linear velocity of the fluid in the secondchannel. In embodiments, the plurality of channels comprise a first anda second channel wherein each of the first and second channels have atop surface and a bottom surface, wherein each channel comprises acoating configured to control liquid flow from a proximal location to adistal location, and the linear velocity of fluid in the first channelis different from the linear velocity of the fluid in the secondchannel. In embodiments, the plurality of channels comprises a firstchannel and a second channel wherein the linear velocity of the liquidin the first channel is equal to the linear velocity of the liquid inthe second channel. In embodiments, the linear velocity of liquid in afirst channel is no less than 10 percent higher than the linear velocityof liquid in a second channel. In embodiments, the linear velocity ofliquid in a first channel is no less than 20 percent higher than thelinear velocity of liquid in a second channel. In embodiments, thelinear velocity of liquid in a first channel is no less than 50 percenthigher than the linear velocity of liquid in a second channel. Inembodiments, the linear velocity of liquid in a first channel is no lessthan 100 percent higher than the linear velocity of liquid in a secondchannel. In embodiments, the linear velocity of liquid in a firstchannel is no less than 200 percent higher than the linear velocity ofliquid in a second channel. In embodiments, the linear velocity ofliquid in a first channel is no less than 300 percent higher than thelinear velocity of liquid in a second channel. In embodiments, thelinear velocity of liquid in a first channel is no less than 500 percenthigher than the linear velocity of liquid in a second channel. Inembodiments, the linear velocity of liquid in a first channel is no lessthan 1000 percent higher than the linear velocity of liquid in a secondchannel. In embodiments, the linear velocity of liquid in a firstchannel is no less than 2000 percent higher than the linear velocity ofliquid in a second channel. In embodiments, the linear velocity ofliquid in a first channel is no less than 5000 percent higher than thelinear velocity of liquid in a second channel. In embodiments, thelinear velocity of liquid in a first channel is no less than 10,000percent higher than the linear velocity of liquid in a second channel.In embodiments, the linear velocity of liquid in a first channel is noless than 50,000 percent higher than the linear velocity of liquid in asecond channel.

In embodiments, the linear velocity of liquid in a first or secondchannel is no more than 0.02 mm/s. In embodiments, the linear velocityof liquid in a first or second channel is no more than 0.1 mm/s. Inembodiments, the linear velocity of liquid in a first or second channelis no more than 1 mm/s. In embodiments, the linear velocity of liquid inthe first or second channel is no more than 5 mm/s. In embodiments, thelinear velocity of liquid in a first or second channel is no more than10 mm/s. In embodiments, the linear velocity of liquid in the first orsecond channel is no more than 15 mm/s. In embodiments, the linearvelocity of liquid in a first or second channel is no more than 20 mm/s.In embodiments, the linear velocity of liquid in the first or secondchannel is no more than 50 mm/s. In embodiments, the linear velocity ofliquid in the first or second channel is no more than 100 mm/s. Inembodiments, the linear velocity of liquid in a first or second channelis no more than 200 mm/s. In embodiments, the linear velocity of liquidin the first or second channel is no more than 300 mm/s. In embodiments,the linear velocity of liquid in a first or second channel is no lessthan 0.005 mm/s. In embodiments, the linear velocity of liquid in thefirst or second channel is no less than 0.01 mm/s. In embodiments, thelinear velocity of liquid in a first or second channel is no less than0.05 mm/s. In embodiments, the linear velocity of liquid in the first orsecond channel is no less than 0.1 mm/s. In embodiments, the linearvelocity of liquid in a first or second channel is no less than 0.5mm/s. In embodiments, the linear velocity of liquid in the first orsecond channel is no less than 1 mm/s. In embodiments, the linearvelocity of liquid in a first or second channel is no less than 5 mm/s.In embodiments, the linear velocity of liquid in the first or secondchannel is no less than 10 mm/s. In embodiments, the linear velocity ofliquid in the first or second channel is no less than 50 mm/s. Inembodiments, the linear velocity of liquid in a first or second channelis no less than 100 mm/s. In embodiments, the linear velocity of liquidin the first or second channel is no less than 200 mm/s.

The coating can be configured with a coating composition that provides asurface energy gradient region on a surface from a proximal location toa distal location wherein the contact angle formed between water and thesurface at a proximal location of the region is different from thecontact angle formed between water and the surface at a distal locationof the region. The coating can be configured so that the contact anglechanges in a continuous fashion or in a step-wise fashion on the surfacefrom a proximal location to a distal location. In embodiments, theproximal location corresponds to the channel entrance, and the distallocation corresponds to the channel exit. In embodiments, the proximallocation corresponds to a location downstream of the channel entrance.In embodiments, the proximal location corresponds to a location on thechannel that is at the mid-point between the channel entrance and thechannel exit. In embodiments, the proximal location corresponds to alocation on the channel that is downstream of the mid-point between thechannel entrance and the channel exit. In embodiments, the proximallocation corresponds to a location on the channel that is upstream ofthe mid-point between the channel entrance and the channel exit. Inembodiments, the distal location corresponds to a location upstream ofthe channel exit. In embodiments, the distal location corresponds to alocation on the channel that is at the mid-point between the channelentrance and the channel exit. In embodiments, the distal locationcorresponds to a location on the channel that is downstream of themid-point between the channel entrance and the channel exit. Inembodiments, the proximal location corresponds to a location on thechannel that is upstream of the mid-point between the channel entranceand the channel exit. In embodiments the distance between the proximallocation and the distal location can be no less than 1 millimeters. Inembodiments the distance between the proximal location and the distallocation can be no less than 3 millimeters. In embodiments the distancebetween the proximal location and the distal location can be no lessthan 5 millimeters. In embodiments the distance between the proximallocation and the distal location can be no less than 10 millimeters. Inembodiments the distance between the proximal location and the distallocation can be no less than 15 millimeters. In embodiments the distancebetween the proximal location and the distal location can be no lessthan 20 millimeters. In embodiments the distance between the proximallocation and the distal location can be no less than 25 millimeters. Inembodiments the distance between the proximal location and the distallocation can be no less than 50 millimeters. In embodiments the distancebetween the proximal location and the distal location can be no lessthan 100 millimeters. In embodiments, the distance between the proximallocation and the distal location can be no more than 0.1 millimeters. Inembodiments, the distance between the proximal location and the distallocation can be no more than 0.5 millimeters. In embodiments, thedistance between the proximal location and the distal location can be nomore than 1 millimeter. In embodiments, the distance between theproximal location and the distal location can be no more than 5millimeters. In embodiments, the distance between the proximal locationand the distal location can be no more than 10 millimeters. Inembodiments, the distance between the proximal location and the distallocation can be no more than 25 millimeters. In embodiments, thedistance between the proximal location and the distal location can be nomore than 50 millimeters. In embodiments, the distance between theproximal location and the distal location can be no more than 200millimeters. In embodiments, the distance between the proximal locationand the distal location can be no more than 500 millimeters.

The composition of the coating as well as the channel dimension can beadjusted based on the particular liquid used and the liquid propertiesincluding viscosity, density, surface tension, and the contact angle theliquid forms with the coating or surface to configure the coating toprovide flow control for many different microfluidic systems using manydifferent fluids. Preferred methods and materials for creating coatingswill vary for many reasons including the substrate used for the surface,the chemical species selected, the surface activation method chosen,cost, fluid solutions selected, and operating conditions for theprocess. The surfaces of the fluid channels can comprise coated anduncoated regions.

Microfluidic products can be created by many known methods includingmachining, micromolding, embossing, additive manufacturing,thermorforming, injection molding, laser-etching, chemical etching,UV-exposure, chemical or physical deposition, etc. These methods canalso be used to create the flow passages and wells configured to allowliquid flow through the device. Components and sub-assemblies of themicrofluidic products can be produced by one or more manufacturingmethod and then combined with other components and sub-assembliesmanufactured by a different method. For the invention many possiblematerials can be used as the material for the microfluidic products ofthe invention; suitable materials include PTFE, polycarbonate,polypropylene, polyethylene, PDMS, polyester, nylon, PMMA, COC polymer,acrylic, glasses, metals, ceramics, etc. The microfluidic products ofthe invention can be fabricated using other different manufacturingmethods, such as photolithography techniques, micromachining technology,or additive manufacturing. Such methods that may be used to fabricatechannels, substrates, and products according to the invention are wellknown in the art and include film deposition processes, such as spincoating and chemical vapor deposition, laser fabrication orphotolithographic techniques, or etching methods, which may be performedeither by wet chemical or plasma processes.

Microfluidic products may be constructed using different manufacturingtechniques. For example, the microfabrication methods used to makemicrochips in the computer industry may also be used to createmicrofluidic products, enabling the creation of intricate, minutepatterns of interconnected channels. Once a pattern is created,microchip manufacturing methods can be employed to recreate the channeldesign on a surface, layer, or component of the microfluidic product. Insome instances, chemical etching or stamping techniques can be employed.As a result, highly precise channels with dimensions that can be variedin their width and depth may be produced. Once the pattern is produced,a cover plate can be affixed or sealed over the surface so as to formconduits in combination with the channels.

Different solid substrate materials may be used in practice of thepresent invention. For example, useful substrates may be opaque,translucent, clear, textured, patterned, rough, smooth, rigid, flexible,treated, primed, or a combination thereof. The substrate typicallycomprises organic and/or inorganic material. The substrate may be, forexample, thermoplastic, thermoset, or a combination thereof. Exemplarysubstrates include films, plates, tapes, rolls, molds, sheets, blocks,molded articles, fabrics, and fiber composites (e.g., circuit boards),and may comprise at least one organic polymer such as polyimide,polyester, acrylic, polyurethane, polyether, polyolefin (e.g.,polyethylene or polypropylene), polyolefin-copolymer, polyimide, andcombinations thereof. Exemplary inorganic substrates include metals(e.g., chromium, aluminum, copper, nickel, silver, gold, steel, andalloys thereof), ceramics, glass, china, quartz, polysilicon, andcombinations thereof. For microfluidic products comprised of laminatedlayers, each layer can comprise a different substrate material. Theproduct can comprise a plurality of layers. In embodiments, theplurality of layers comprises a first layer and a second layer whereinthe first layer comprises a different polymer than the second layer. Inembodiments, the plurality of layers comprises a first layer and asecond layer wherein the first layer comprises the same polymer as thesecond layer. In embodiments, the fluid passages of the microfluidicproducts can be located at any position within the cartridge andoriented at any angle. In an embodiment, the fluid passages are located,primarily, in planar networks, located proximate to the outside surfacesto allow for a multi-layered cartridge design that uses, e.g., machined,die-cut, laser-cut and/or molded cartridge body component. Inembodiments, fluid passage geometries include passages withcross-sections that are circular, oval, square or rectangular incross-section. Width and height can vary widely from nm to cm rangesdepending on the application, sample volume and cartridge design. Rangesfor the height are 0.02 to 2 mm, more preferably, 0.05 to 1.5 mm, mostpreferably 0.05 mm to 1 mm.

The fluidic network may be formed within the cartridge in a number ofdifferent ways, dependent, in part, upon the materials chosen for thecartridge. Any known fabrication method appropriate to the cartridgebody material may be employed including, but not limited to,stereolithography, chemical/laser etching, integral molding, machining,lamination, etc. Such fabrication methods may be used alone or incombination. In certain embodiments of the invention, the cartridgecomprises a cartridge body and one or more cover layers mated tosurfaces of the cartridge body so as to define one or more fluidicnetworks preferably, planar fluidic networks) therebetween. Similarly,z-transitions and/or ports can be selectively molded into, or machinedout of, the cartridge body at predetermined locations to form thefluidic connections between the fluid passages on the upper and lowersurfaces.

One embodiment of the cartridge may be fabricated using a “lamination”process whereby the cartridge body's functional surfaces are sealedusing cover layers to form the fluidic network. For example, recesses(e.g., channels, grooves, wells, etc.) can be manufactured into one ormore surfaces of the cartridge body to provide a recessed pattern of thefluidic network. Sealing/mating of the recessed patterns to cover layersforms a fluidic network comprising fluidic components (e.g., conduits,chambers, etc.) at least some of which are defined in part by therecesses in the cartridge body and in part by a surface of a coverlayer. In an embodiment, the cover layers are comprised of plastic film.The cover layer may be coated with an adhesive to seal the cover layeragainst the cartridge layer. Other methods for mating the cover layer tothe cartridge body will be known to the skilled artisan, e.g., the sealmay be achieved by heat sealing, ultrasonic welding, RF (radiofrequency) welding, by solvent welding (applying a solvent between thecomponents that softens or partially dissolves one or both surfaces), byuse of an intervening adhesive layer (e.g., a double sided adhesivetape, etc.). Advantageously, cartridge features that are created bypatterned deposition (e.g., patterned deposition of electrode ordielectric layers and/or patterned deposition of reagents to form dryreagent pills or to form binding domains with immobilized bindingreagents) are created on cover layers so as to take advantage ofautomation available to process plastic film in large sheets or rolls.

Recesses may be, e.g., molded in, etched in or machined from thecartridge body. By analogy, fluidic components may also be defined, atleast in part, by recesses in a cover layer that is mated to a cartridgebody. Fluidic components may also be defined, at least in part, byregions cutout from gasket layers disposed between the cartridge bodyand cover layers. Apertures in the cartridge body and/or cover layersmay be used to provide for access ports to the fluidic network, e.g.,sample introduction ports, vent ports, reagent addition ports and thelike. Vent ports can be used to allow the equilibration of fluid in thechambers with the atmosphere or to allow for the directed movement offluid into or out of a specified chamber by the application of positiveor negative pressure. Vent ports are designed to prevent the leakage ofliquid samples or reagents through the ports and may includeaerosol-resistance filters, membrane or filter materials that permit airflow but act as barriers to liquid solutions and materials that areporous to air but seal when they come in contact with solutions.

The products of the invention can comprise a surface comprising a glass,metal, metal oxide, or polymer surface. In an embodiment, the surface isa metal oxide comprising a metal oxide from the group comprising silica,alumina, quartz, glass, or the like and the coating configured tocontrol liquid flow comprises carboxylic acid moiety. In an embodiment,the base surface is a metal selected from the group comprising gold,silver, copper, cadmium, zinc, palladium, platinum, mercury, lead, iron,chromium, manganese, tungsten, and any of their alloys, and the coatingcomprises a sulfur-containing moiety (e.g.thiols, sulfides, disulfides,and the like). In another embodiment, the surface is doped or undopedsilicon and the coating comprises a silane or chlorosilane species. Inanother embodiment, the surface is a metal selected from the groupcomprising palladium and platinum and the coating comprises a nitritesor isonitrile species. In an embodiment, the surface is copper and thecoating comprises a hydroxamic acid species. In another embodiment, thesurface is gold and the coating comprises at least one sulfur-containingfunctional group selected from the group comprising thiols, sulfides, ordisulfides. In an embodiment, the product of the invention comprises asurface comprising a cylic olefin copolymer. In an embodiment, themicrofluidic product of the invention comprises a surface comprising adielectric material.

In embodiments, the surfaces of the fluid passages can comprisepolymeric species selected to exhibit one or more properties desired forthe surface or other substrate to which the polymer molecules arebonded. In embodiments, the coating configured to control liquid flow inthe microfluidic product can comprise polymeric species selected toexhibit one or more properties desired for the surface or othersubstrate to which the polymer molecules are bonded. For example, it maybe desired in some instances to provide polymeric species with veryhydrophilic properties, in which case polymer species such as hyaluronicacid may be employed. The polymer polyethylene glycol may be employed torepel proteins from a surface. Heparin, a polysaccharide, may be used toimpart antithrombogenic characteristics, and chitosan may be employed toprovide hemostatic properties. In another embodiment, the polymerspecies comprises ionic, nonionic, polar, nonpolar, halogenated, alkyl,aryl or other functionalities.

In embodiments, the compounds used to form the coating compositions forthe coating configured to control liquid flow can have the generalformula X-J-M where X represents a species that forms the bond to thesurface, J represents a spacer moiety or polymer backbone species, and Mrepresents a functional group that is provided to the surface of thecoating.

Species X1, X2, . . . Xn can be selected based on the surface materialsand bonding requirements desired. Species J1, J2, . . . Jn can beselected based on the properties of the polymer chain desired, includingchain length, film stability, cross-linking capabilities, reactivity,etc. Species M1, M2, . . . Mn can be selected based on the surfaceenergy properties desired as well as other functional properties desiredincluding reactivity, adsorption, bonding, etc. In embodiments, multiplen solutions comprising compounds of Xn-Jn-Mn can be used. Inembodiments, X-J-M compounds can form self-assembled monolayers fromsolution.

In embodiments, the functional group M1, M2, . . . Mn is selected fromthe group comprising ionic, nonionic, polar, nonpolar, halogenated,alkyl, aryl or other functionalities,

In other embodiments, the functional group M1, M2, . . . Mn can includeany one of the following: —OH, —CONHR, —CONHCOR, —NHR, —COOH, —COOR,—CSNHR, —COR, —RCSR, —RSR, —ROR, —SOOR, —RSOR, —CONR₂, —(OCH₂CH₂)_(n)OH, —(OCH₂ CH₂)_(n)OR—CH₃, —NR₂, —CN, —(CF₂)_(n)CF₃, —CO₂CH₃,—CONHCH₃, —CR, CHCH₂, —OCH₂CF₂CF₃, Cl, Br, olefins, and the like, andany combination thereof.

In the above list, R is hydrogen or an organic group such as ahydrocarbon or fluorinated hydrocarbon. As used herein, the term“hydrocarbon” includes alkyl, alkenyl, alkynyl, cycloalkyl, aryl,alkaryl, aralkyl, and the like. The hydrocarbon group may, for example,comprise methyl, propenyl, ethynyl, cyclohexyl, phenyl, tolyl, andbenzyl groups. The term “fluorinated hydrocarbon” is meant to refer tofluorinated derivatives of the above-described hydrocarbon groups.

In another embodiment, J is a hydrocarbon chain with theformula—(CH₂)_(n)—where n is between 1 and 22, preferably between 2 and18, more preferably between 2 and 12. In some embodiments using metaloxide base surfaces, the functional group X is a carboxylic acid.

In an additional embodiment, the base surface is a metal selected fromthe group comprising gold, silver, copper, aluminum, cadmium, zinc,palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten,and any alloys of the above. In some embodiments using metals for thebase surfaces, the functional group X is a sulfur-containing functionalgroup (e.g.thiols, sulfides, disulfides, and the like). In otherembodiments, the metal of the base surface is in the form of a metalizedfilm coating a polymer surface.

In another embodiment, the base surface is doped or undoped silicon. Insome embodiments using doped or undoped silicon for the base surface,the functional group X is selected from the group comprising silanes orchlorosilanes. In another embodiment, the base surface is a metalselected from the group comprising palladium and platinum. In someembodiments using these metals for the base surface, the functionalgroup X is a functional group selected from the group comprisingnitrites and isonitriles. In another embodiment, the base surface iscopper. In some embodiments using copper for the base surface, thefunctional group X is a hydroxamic acid. In another embodiment, the basesurface is gold. In some embodiments using gold for the base surface,the functional group X is at least one sulfur-containing functionalgroup selected from the group consisting of thiols, sulfides, ordisulfides.

In one embodiment a method of derivatizing a surface with a mixedmonolayer to create a surface energy gradient comprises the followingsteps:

a) exposing a base surface having a proximal and a distal location to afirst solution comprising a plurality of molecules of the formulaX1-J1-M1, wherein X1 and M1 represent separate functional groups and J1represents a spacer moiety that, together, are able to promote formationfrom solution of a self-assembled monolayer for sufficient time to forma monolayer surface having a substantially uniform surface energy on thebase surface,

b) removing a portion of the monolayer of step (a) such that a portionof the base surface is again fully or partially exposed,

c) exposing the portion of the base surface from (b) to a secondsolution comprising a plurality of molecules of the formula X2-J2-M2 anda plurality of molecules of the formula X1-J1-M1 wherein the functionalgroup M2 has a different surface energy from that of the functionalgroup M1 such that a surface energy gradient from a proximal location toa distal location is formed. The X2 and J2 groups for the molecule inthe second solution can be the same as the X1 and J1 groups for themolecule in the first solution, or they can be different, depending onthe desired final properties of the mixed monolayer.

In some preferred embodiments, at least one of the molecules of formula(X-J-M) chosen to form the coating configured to control liquid flow isresistant to the adsorption of biopolymers such as proteins, enzymes,antibodies, polynucleic acids, cells, and other biological molecules. Bythe term “resistant to the adsorption of biopolymers” it is meant thatthe base surface covered by the coating has a reduction in the amount ofa biopolymer adsorbed on the surface, when contacted with a mediumcontaining biopolymers available for adsorption, as compared to theamount adsorbed on the same base surface that is not covered by thecoating. In some embodiments, the coating configured to control liquidflow is a monolayer.

For some embodiments, the J group of the molecule is a spacer moietycomprising a biopolymer-resistant domain. Suitable moieties for thebiopolymer-resistant domain of the J group are discussed in U.S. Pat.No. 6,235,340 and include oligoethers, oligoglycols, oligoalcohols,oligocarbonyls, oligosulfides, oligosulfones and oligosaccharides. Suchmoieties typically are used to produce a monolayer or other coating thatis both hydrophilic and biopolymer-resistant.

In one embodiment, the biopolymer-resistant domain comprises anoligo-(ethylene glycol) linkage (—OCH₂CH₂-)_(n) where n is 2 to 4.

The surfaces of the fluid passages or the coating configured to controlliquid flow may use polymers that are natural or synthetic in origin.Such polymers include oligomers, homopolymers and copolymers resultingfrom addition or condensation polymerization, and natural polymersincluding oligosaccharides, polysaccharides, peptides, and proteins. Thepolymers may include several distinct polymer types, as prepared byterminal or side chain grafting The polymers of the invention mayinclude cellulose-based products such as hydroxyethyl cellulose,hydroxypropyl cellulose, carboxymethyl cellulose, cellulose acetate andcellulose butyrate, acrylics such as those polymerized from hydroxyethylacrylate, hydroxyethyl methacrylate, glyceryl acrylate, glycerylmethacrylate, acrylic acid, methacrylic acid, acrylamide andmethacrylamide, vinyls such as polyvinyl pyrrolidone and polyvinylalcohol, nylons such as polycaprolactam, polylauryl lactam,polyhexamethylene adipamide and polyhexamethylene dodecanediamide,polyurethanes, polylactic acids, linear polysaccharides such as amylose,dextran, chitosan, and hyaluronic acid, and branched polysaccharidessuch as amylopectin, hyaluronic acid and hemi-celluloses.

In an embodiment, the surfaces of the fluid passages can comprise latentreactive (e.g., photoreactive) groups bonded to the surface itself. Inan embodiment, the coating configured to control liquid flow cancomprise latent reactive (e.g., photoreactive) groups bonded to thesurface itself. For instance, with ceramic or glass surfaces, aphotoreactive silane can be used. Similarly, with surfaces of gold orother noble metals, an intermediate layer can be provided using aphotoreactive sulfur compound (e.g., thiol or thioether such as methylthioxanthone) or other suitable compound. In another embodiment, a SAM(self-assembled monolayer) can be formed at a suitable interface, andoptionally transferred to a solid support surface. The surface, in turn,can be provided by a material selected from ceramics, metals andpolymeric materials. For instance, the surface can be provided by amaterial selected from organosilane-pretreated glasses,organosilane-pretreated silicon materials, and silicon hydrides, or by apolymeric material selected from the group consisting of polystyrene,polycarbonate, polyester, polyethylene, polyethylene terephthalate(PET), polyglycolic acid (PGA), polyolefin,poly-(p-phenyleneterephthalamide), polyphosphazene, polypropylene,polytetrafluoroethylene, polyurethane, polyvinyl chloride, polyacrylate(including polymethacrylate), and silicone elastomers, as well ascopolymers and combinations thereof.

The surfaces of the fluid passages and/or the coating configured tocontrol liquid flow can comprise a photoreactive group. Photoreactivegroups respond to specific applied external stimuli to undergo activespecie generation with resultant covalent bonding to an adjacentchemical structure, e.g., as provided by the same or a differentmolecule. Photoreactive groups are those groups of atoms in a moleculethat retain their covalent bonds unchanged under conditions of storagebut that, upon activation by an external energy source, form covalentbonds with other molecules. Upon activation of the photoreactive groups,the reagent molecules are covalently bound to each other and/or to thematerial surface by covalent bonds through residues of the photoreactivegroups. The photoreactive groups generate active species such as freeradicals and particularly nitrenes, carbenes, and excited states ofketones upon absorption of electromagnetic energy. Photoreactive groupsmay be chosen to be responsive to various portions of theelectromagnetic spectrum, and photoreactive groups that are responsiveto e.g., ultraviolet and visible portions of the spectrum are preferredand may be referred to herein occasionally as “photochemical group” or“photogroup”. Latent reactive groups can be chosen that are responsiveto various portions of the electromagnetic spectrum, with thoseresponsive to ultraviolet and visible portions of the spectrum (referredto herein as “photoreactive”) being particularly preferred. In anembodiment, the coating can provide latent reactive groups to thesurface, for instance, wherein the surface comprises a ceramic, siliconoxide, metal oxide, or glass surface, and the coating comprises aphotoreactive silane.

Photoreactive aryl ketones, such as acetophenone, benzophenone,anthraquinone, anthrone, and anthrone-like heterocycles (i.e.,heterocyclic analogs of anthrone such as those having N, O, or S in the10-position), or their substituted (e.g., ring substituted) derivativesmay be used in the coating configured to control liquid flow. Thefunctional groups of such ketones are readily capable of undergoing theactivation/inactivation/reactivation cycle described herein.Benzophenone is a preferred photoreactive moiety, since it is capable ofphotochemical excitation with the initial formation of an excitedsinglet state that undergoes intersystem crossing to the triplet state.The excited triplet state can insert into carbon-hydrogen bonds byabstraction of a hydrogen atom (from a support surface, for example),thus creating a radical pair. Subsequent collapse of the radical pairleads to formation of a new carbon-carbon bond. If a reactive bond(e.g., carbon-hydrogen) is not available for bonding, the ultravioletlight-induced excitation of the benzophenone group is reversible and themolecule returns to ground state energy level upon removal of the energysource. In an embodiment, the surface comprises a polymer surface andthe coating comprises a photoreactive aryl ketone. Additionalphotoreactive groups include azides. The azides constitute a class ofphotoreactive groups and include arylazides such as phenyl azide andparticularly 4-fluoro-3-nitrophenyl azide, acyl azides such as benzoylazide and p-methylbenzoyl azide, azido formates such as ethylazidoformate, phenyl azidoformate, sulfonyl azides such asbenzenesulfonyl azide, and phosphoryl azides such as diphenyl phosphorylazide and diethyl phosphoryl azide. Diazo compounds constitute anotherclass of photoreactive groups and include diazoalkanes such asdiazomethane and diphenyldiazomethane, diazoketones such asdiazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone,diazoacetates such as t-butyl diazoacetate and phenyl diazoacetate, andbeta-keto-alpha-diazoacetates such as t-butyl alpha diazoacetoacetate.Other photoreactive groups include the diazirines such as3-trifluoromethyl-3-phenyldiazirine, and ketenes such as ketene anddiphenylketene.

The surfaces of the fluid passages and/or the coatings configured tocontrol liquid flow may contain one or more thermochemically reactivegroups (i.e., groups having a reaction rate dependent on temperature).Suitable groups are selected from the group consisting of activatedesters, epoxide, azlactone, activated hydroxyl and maleimide groups.Those skilled in the art would also recognize numerous otheramine-reactive functional groups such as isocyanates, thioisocyanates,carboxylic acid chlorides, epoxides, aldehydes, alkyl halides andsulfonate esters, such as mesylate, tosylate and tresylate, each ofwhich could serve as the thermochemically reactive group. Optionally,the coating can also contain one or more photoreactive groups.Additionally, the coating may comprise one or more hydrophilic polymers,to which the thermochemically reactive and/or photoreactive groups canbe pendent. The photoreactive groups (alternatively referred to hereinas “photogroups”) can be used, for instance, to attach molecules to thesurface of the support upon the application of a suitable energy sourcesuch as light. The thermochemically reactive groups, in turn, can beused to form covalent bonds with appropriate and complementaryfunctional groups on a different molecule. In another embodiment, thecoating can comprise self-assembling monolayer molecules wherein theself-assembling monolayer molecules themselves provide thermochemicalreactive groups and the method comprises the further step of attachingbinding molecules to the monolayer by reaction between correspondingreactive groups of the binding molecules and the reactive groups of theself-assembling monolayer molecules.

Polymers appropriate for use as either a surface or component of themicrofluidic product include a variety of biocompatible polymers knownin the art to be suitable for use in life science applications. Thebiocompatible polymer may be biostable or biodisintegrable. By“biostable” is meant a polymer that does not substantially disintegrate(i.e., deteriorate) in vivo. Thus, a biostable polymer is one thatmaintains its structural integrity, i.e., is substantially inert, in thepresence of a physiological environment. “Biodisintegrable” polymers arethose that undergo substantial deterioration in vivo, and includesoluble polymers, bioerodable polymers and biodegradable polymers.

Biocompatible biostable polymers include numerous thermoplastic andelastomeric polymeric materials that are known in the art. Polyolefinssuch as metallocene catalyzed polyethylenes, polypropylenes, andpolybutylenes and copolymers thereof, ethylenic polymers such aspolystyrene; ethylenic copolymers such as ethylene vinyl acetate (EVA),ethylene-methacrylic acid and ethylene-acrylic acid copolymers wheresome of the acid groups have been neutralized with either zinc or sodiumions (commonly known as ionomers); polyacetals; chloropolymers such aspolyvinylchloride (PVC); fluoropolymers such as polytetrafluoroethylene(PTFE); polyesters such as polyethylene terephthalate (PET);polyester-ethers; polysulfones; polyamides such as nylon 6 and nylon6,6; polyamide ethers; polyethers; elastomers such as elastomericpolyurethanes and polyurethane copolymers; silicones; polycarbonates;and mixtures and block or random copolymers of any of the foregoing arenon-limiting examples of biostable biocompatible polymers useful formanufacturing the medical devices of the present invention.

Additional polymers that can be used as surfaces or components of themicrofluidic product include polyurethanes, silicones,poly(meth)acrylates, polyesters, polyalkylene oxides such aspolyethylene oxide, polyvinyl alcohols, polyethylene glycols andpolyvinyl pyrrolidone; hydrogels such as those formed from crosslinkedpolyvinyl pyrrolidone and polyesters could also be used. Other polymersinclude polyolefins, polyisobutylene and ethylene-alphaolefincopolymers; acrylic polymers (including methacrylic polymers) andcopolymers, vinyl halide polymers and copolymers, such as polyvinylchloride; polyvinyl ethers, such as polyvinyl methyl ether;polyvinylidene halides such as polyvinylidene fluoride andpolyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinylaromatics such as polystyrene; polyvinyl esters such as polyvinylacetate; copolymers of vinyl monomers with each other and olefins, suchas ethylene-methyl methacrylate copolymers, acrylonitrile-styrenecopolymers, ABS resins and ethylene-vinyl acetate copolymers;polyamides, such as nylon 6,6 and polycaprolactam; alkyd resins;polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins;rayon; rayon-triacetate, cellulose, cellulose acetate, cellulose acetatebutyrate; cellophane; cellulose nitrate; cellulose propionate; celluloseethers (i.e. carboxymethyl cellulose and hydroxyalkyl celluloses); andcombinations thereof. Mixtures and block or random copolymers of any ofthe foregoing are also useful in the present invention.

In addition to being used as surfaces or components in the microfluidicproduct, biodisintegrable polymers can also be incorporated into thecoating configured to control liquid flow in the microfluidic product.Biodisintegrable polymers include, but are not limited to, polylacticacid, polyglycolic acid and copolymers and mixtures thereof such aspoly(L-lactide) (PLLA), poly(D,L-lactide), polyglycolic acid(polyglycolide), poly(L-lactide-co-D,L-lactide),poly(L-lactide-co-glycolide), poly(D, L-lactide-co-glycolide),poly(glycolide-co-trimethylene carbonate),poly(D,L-lactide-co-caprolactone), poly(glycolide-co-caprolactone),polyethylene oxide (PEO), polydioxanone, polypropylene fumarate,poly(ethyl glutamate-co-glutamic acid),poly(tert-butyloxy-carbonylmethyl glutamate), polycaprolactone,polycaprolactone co-butylacrylate, polyhydroxybutyrate and copolymers ofpolyhydroxybutyrate, poly(phosphazene), poly(phosphate ester),poly(amino acid) and poly(hydroxy butyrate), polydepsipeptides, maleicanhydride copolymers, polyphosphazenes, polyiminocarbonates, poly[(97.5%dimethyl-trimethylene carbonate)-co-(2.5% trimethylene carbonate)],cyanoacrylate, hydroxypropylmethylcellulose, polysaccharides such ashyaluronic acid, chitosan and regenerate cellulose, tyrosine-basedpolymers (e.g., tyrosine-derived polycarbonates such as the Tyrosorb™Synthetic Polymers available from Integra LifeSciences and thosedescribed in U.S. Pat. No. 6,120,491), and proteins such as gelatin andcollagen and genetically engineered variants thereof (e.g., collagenengineered to include thrombin cleavage sites), as well as mixtures andcopolymers of the above, among others. Additional polymers includealiphatic polyesters, poly(amino acids), copoly(ether-esters),polyalkylene oxalates, polyamides, poly(iminocarbonates),polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesterscontaining amido groups, poly(anhydrides), polyphosphazenes,biomolecules, and blends thereof. Aliphatic polyesters includehomopolymers and copolymers of lactide (which includes lactic acid d-,l-and meso lactide), epsilon-caprolactone, glycolide (including glycolicacid), hydroxybutyrate, hydroxyvalerate, para-dioxanone, trimethylenecarbonate (and its alkyl derivatives), 1,4-dioxepan-2-one,1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one and polymer blendsthereof. Biodisintegrable polymers also include naturally occurringmaterials that may be enzymatically degraded in the human body or arehydrolytically unstable in the human body such as fibrin, fibrinogen,collagen, elastin, and absorbable biocompatible polysaccharides such aschitosan, starch, fatty acids (and esters thereof), glucoso-glycans andhyaluronic acid. Mixtures and block or random copolymers of any of theforegoing are also contemplated.

Gradient coatings can be applied to the surfaces using differentdispensing and coating applications. Several manufacturers includingEpson, Hewlett Packard, Biofluidix, BioDot, Agilent, Life Technologies,Formulatrix, Deerac, and others manufacture systems that can dispensesub-microliter droplets with precision and accuracy to produce one ormore products of the invention. U.S. Pat. No. 7,790,265 and US2003/0049381 (dip-pen nanolithography) disclose methods for using AFMtips to pattern surfaces. In an embodiment, the coating may be formed onthe surface using an AFM or dip-pen nanolithography.

In an embodiment, the microfluidic product comprises a channel surfacecomprising a surface energy gradient coating wherein the surface is alsoconfigured for electrowetting or heating. In this embodiment, thesurface energy gradient coating may reduce the electrical or thermalenergy required to move liquids through the channel.

A surface with one or more surface energy gradient regions surrounded byregions of uncoated or uniformly-coated surfaces can be used in theinvention. In one embodiment, the starting surface is a uniform surfacethat already has a monolayer film on it. The uniform monolayer filmcould be removed using a laser or a solvent or chemical etch. Thegradient could be created on the newly exposed regions using any of themethods previously described in the prior art. In another embodiment,the surface energy gradient regions are created first using the methodsdescribed in the prior art. Then the uncoated areas of the surface couldbe coated with a uniform monolayer to create the desired surface.

Multiple manufacturing steps can be used to manufacture the channelsconfigured to control liquid flow in the microfluidic products of thisinvention. The channels can vary in length, width, and height. In anembodiment, a different solution or even air or other gas could be usedto displace solutions from the channel. In an embodiment, different flowpaths with different entrances and exits for the fluids can be used tocreate surface energy gradients in different directions. In anembodiment, mixers can be added to improve the mixing of the solutionsbefore they are delivered to the channel to apply the coating. In anembodiment, different solutions could be used with different reactivespecies and/or concentrations to produce different types of surfaceenergy gradients. In an embodiment, a plurality of surface energygradients could be produced on a plurality of flow paths, includingradial flow out from a point, multiple parallel channels out from acentral axis, and any other pattern that can be manufactured. Inembodiments, methods for preparing the surface can include simplecleaning, acid activation, electrochemical activation, UV-activation,chemical activation, etc.

Microfluidic products of the invention can be used in many differentproduct applications. The microfluidic products of the invention can beused with many different fluids, including bodily fluids commonly usedfor diagnostic testing. These bodily fluids include blood, tears,saliva, plasma, urine, sweat, and others. In an embodiment, themicrofluidic product is used with a solution comprising a therapeutic ortreatment agent. In an embodiment, the microfluidic product is used withanalytical buffer solutions.

The microfluidic product may comprise a microscope coverslip, microscopeslide, petri dish, tissue culture flask, biomedical implant, diagnosticassay, a biochip, a protein/nucleic acid biochip sensor, a cell-basedsensor, a lab-on-a-chip assay, a lab-in-a-capillary assay, a celladhesion assay, a cell translocation/migration/invasion/chemotaxisassay, or a neuronal-guidance assay. The microfluidic product maycomprise a non-flat substrate. The microfluidic product may have anexterior side opposite an interior side, and wherein the interior sidehas the coating applied thereon. The substrate may include silicon,plastic, rubber, metal, ceramic material.

Life science applications where the microfluidic products can be usedinclude in-vitro diagnostic devices, lab-on-a-chip devices, microarrays,microplates, analytical slides, organ-on-a-chip, system-on-a-chip, andother diagnostic products. The microfluidic products can also be used inmedical devices for fluid transport or drainage, or for drug delivery.These medical devices include implantable devices such as stents,shunts, insulin pumps, ports, and catheters and others. The microfluidicproducts can also be used in wound healing devices or patches. In anembodiment, the invention provides surface modification and enhancementof microarrays for sequencing. A DNA microarray (also commonly known asDNA chip or biochip) is a collection of microscopic DNA spots attachedto a solid surface. Scientists use DNA microarrays to measure theexpression levels of large numbers of genes simultaneously or togenotype multiple regions of a genome. In various embodiments, themicrofluidic products of the invention can provide the followingbenefits in applications:

-   -   Equality and uniformity in the sample across the target surface,        providing equal signal strength increasing the signal to noise        ratio and improved CV data.    -   Lower volume required of reagent thus saving considerable money        in chemistry usage.    -   Increased density due to the process of coating small landing        pad zones reagent chemistries with a higher density than what        could be achieved with standard screening and or fluid        dispensing.    -   Moving fluids in specific patterns in series and parallel to        enable faster sample reaction times.

In one embodiment of the invention, a microfluidic product comprises atleast one inlet port in communication with a first channel, a detectionregion within fluid communication of the first channel, and a detectorassociated with the detection region. In an embodiment according to theinvention, the microfluidic product comprises a detection region along achannel. There may be a plurality of detection regions and detectors,working independently or together, e.g., to analyze one or moreproperties of a chemical such as a reagent.

The surfaces of the fluid passages and/or the coating configured tocontrol liquid flows can comprise coating compositions that can be usedto form a coating for a variety of medical devices for which it isdesired to provide a functional coating at a surface thereof. Exemplarymedical devices include catheters, other vascular devices (e.g., grafts,valves, artificial hearts, heart assist devices); implantabledefibrillators; blood oxygenator devices (e.g., tubing, membranes);surgical; membranes; cell culture devices; chromatographic supportmaterials; biosensors; shunts for hydrocephalus; wound managementdevices; endoscopic devices; infection control devices; urologicaldevices; colostomy bag attachment devices; ophthalmic devices; glaucomadrain shunts; intraocular lenses; respiratory, peripheralcardiovascular, spinal, neurological, dental, ear/nose/throat (e.g., eardrainage tubes); renal devices; and dialysis (e.g., tubing, membranes,grafts). Other medical devices can include urinary catheters,intravenous catheters, small diameter grafts, vascular grafts,artificial lung catheters, glucose sensors (long-term and short-term),degradable coronary stents (e.g., degradable, non-degradable,peripheral), blood pressure and stent graft catheters, birth controldevices, implanted drug infusion tubes, intravitreal drug deliverydevices, nerve regeneration conduits, oncological implants, painmanagement implants, spinal/orthopedic repair devices, wound dressings,embolic protection filters, heart valves (e.g., mechanical, polymeric,tissue, percutaneous, carbon, sewing cuff), valve annuloplasty devices,mitral valve repair devices, vascular intervention devices, leftventricle assist devices, neurological catheters, left atrial appendagefilters, hemodialysis devices, vascular access catheters, cardiacsensors, uterine bleeding patches, urological catheters/stents/implants,in vitro diagnostics, aneurysm exclusion devices, and neuropatches.Other medical devices include, but are not limited to, vena cavafilters, drug infusion catheters, esophageal stents, circulatory supportsystems, angiographic catheters, transition sheaths and dialators,coronary and peripheral guidewires, hemodialysis catheters,neurovascular balloon catheters, tympanostomy vent tubes, cerebro-spinalfluid shunts, drainage tubes, thoracic cavity suction drainagecatheters, electrophysiology catheters, stroke therapy catheters,abscess drainage catheters, biliary drainage products, dialysiscatheters, central venous access catheters, and parental feedingcatheters. Other medical devices suitable for the present disclosureinclude, but are not limited to implantable vascular access ports,vascular stents, blood tubing, vascular grafts, total artificial heartsand ventricular assist pumps, extracorporeal devices such as bloodoxygenators, blood filters, hemodialysis molecules, hemoperfusionmolecules, plasmapheresis molecules, and hybrid artificial organs suchas pancreas or liver and artificial lungs.

The microfluidic products of the invention can also be used inindustrial applications such as cooling and microreactor applications.Such applications include cooling of electronic devices such as servers,computer chips, and integrated circuits. Typical fluids in theseapplications include water, glycols, alcohols, organic solvents, andaqueous solutions. In an embodiment, the liquid used in a microfluidicproduct of the invention comprises a phase-change material. In anembodiment, the liquid used in the microfluidic product of the inventioncomprises and emulsion. In an embodiment, the liquid used in themicrofluidic product comprises an emulsion comprising nanoparticles. Inan embodiment, the liquid used in the microfluidic product comprises awater-in-oil emulsion or an oil-in-water emulsion. In an embodiment, thefluid used in the microfluidic product comprises a two-phase fluid.

The microfluidic products of the invention can be used with manydifferent liquids or fluids. In an embodiment, surface tension values offluids used with the invention range from 10-150 dynes/cm. In anembodiment, viscosity ranges for the fluid range from 0.1-500centipoise. In certain embodiments, the microfluidic product is designedto only allow for flow of a particular liquid when the liquid attains atargeted viscosity or a targeted surface tension, such as after beingheated or being mixed with another material. In an embodiment, themicrofluidic product can transport adhesive or another sealing materialto a location for bonding materials or sealing leaks. In an embodiment,the microfluidic product can transport a phase-change material.

In an embodiment, the products of the invention can be used with atleast one of the following liquids or mixtures of liquids:

Specific Surface Density Tension Viscosity Viscosity Molecular Molecular(g/ml (dynes/cm @25 C. @25 C. Name Formula Wt @25 C.) @25 C.) (cP) (cs)Bromine Br₂ 159.81 3.214 41 0.94 0.29 Tetrachloromethane CCl₄ 153.821.583 26.3 0.91 0.57 (carbon tetrachloride) Trichloromethane CHCl₃119.38 1.48 26.7 0.54 0.36 (chloroform) Dichloromethane CH₂Cl₂ 84.931.318 27.8 0.41 0.31 (methylene chloride, DCM) Diiodomethane CH₂I₂267.84 3.306 50.8 2.6 0.78 (methylene iodide) Methanoic acid CH₂O₂ 46.031.214 37.7 1.61 1.32 (formic acid) Formamide CH₃NO 45.04 1.129 57 3.342.96 (methanomide) Nitromethane CH₃NO₂ 61.04 1.129 36.3 0.63 0.56Methanol CH₄O 32.04 0.787 22.1 0.54 0.69 (methyl alcohol)Trichloroethylene (TCE, C₂HCl₃ 131.39 1.458 28.7 0.55 0.38trichloroethene) 1,1,1-Trichloroethane C₂H₃Cl₃ 133.4 1.33 25 0.79 0.59(methyl chloroform) Acetonitrile C₂H₃N 41.05 0.779 28.7 0.37 0.47(ethane nitrile) 1,2-Dichloroethane C₂H₄Cl₂ 98.96 1.246 32.6 0.78 0.63(ethylene dichloride) Acetic acid C₂H₄O₂ 60.05 1.043 27 1.06 1.02(ethanoic acid) Ethanol (ethyl alcohol) C₂H₆O 46.07 0.787 22 1.07 1.36Dimethyl sulfoxide C₂H₆OS 78.13 1.095 42.9 1.99 1.82 (DMSO) Ethyleneglycol C₂H₆O₂ 62.07 1.111 48.4 16.1 14.5 2-Aminoethanol C₂H₇NO 61.081.014 48.3 21.1 20.8 (ethanolamine) Acrylonitrile C₃H₃N 53.06 0.801 26.70.34 0.42 (ethenylnitrile) Epichlorohydrin C₃H₅ClO 92.52 1.174 36.3 1.070.91 (chloromethyloxirane) Acetone (propanone) C₃H₆O 58.08 0.786 23 0.310.39 Methyl acetate C₃H₆O₂ 74.08 0.927 24.5 0.36 0.39N,N-Dimethylformamide C₃H₇NO 73.09 0.945 34.4 0.79 0.84 (DMF) 1-Propanol(n-propanol, C₃H₈O 60.1 0.802 20.9 1.95 2.43 n-propyl alcohol)2-Propanol C₃H₈O 60.1 0.783 23.3 2.04 2.61 (isopropyl alcohol)2-Methoxyethanol C₃H₈O₂ 76.1 0.96 42.8 1.7 1.8 (ethylene glycolmonomethyl ether) Dimethoxymethane C₃H₈O₂ 76.1 0.854 18.8 0.33 0.39(methylal) 1,2-Propanediol C₃H₈O₂ 76.1 1.033 45.6 40.4 39.1 (propyleneglycol) Glycerol C₃H₈O₃ 92.09 1.257 76.2 934 743 Propylene carbonateC₄H₆O₃ 102.09 1.200 40.9 2.50 2.08 2-Butanone (methyl C₄H₈O 72.11 0.79924 0.41 0.51 ethyl ketone) Tetrahydrofuran C₄H₈O 72.11 0.88 26.7 0.460.52 1,4-Dioxane C₄H₈O₂ 88.11 1.029 32.9 1.18 1.15 Ethyl acetate C₄H₈O₂88.11 0.894 23.2 0.42 0.47 (ethyl ethanoate) Morpholine C₄H₉NO 87.120.997 38.8 2.02 2.03 1-Butanol C₄H₁₀O 74.12 0.806 25 2.54 3.15 (n-butylalcohol) 2-Butanol C₄H₁₀O 74.12 0.805 22.6 3.1 3.85 (sec-butanol)Diethyl ether C₄H₁₀O 74.12 0.708 16.7 0.22 0.31 (ethoxyethane)2-Methylpropyl alcohol C₄H₁₀O 74.12 0.797 22.6 3.95 4.96 (isobutanol)1,3-Butanediol C₄H₁₀O₂ 90.12 1.002 47.1 98.3 98.1 1,2-DimethoxyethaneC₄H₁₀O₂ 90.12 0.865 20 1.1 1.3 (DME (ethylene glycol dimethyl ether)2-Ethoxyethanol C₄H₁₀O₂ 90.12 0.925 28.8 2.1 2.3 (ethylene glycolmonoethyl ether) Diethylene glycol C₄H₁₀O₃ 106.12 1.114 55.1 30.2 27.1Dimethylethanolamine C₄H₁₁NO 89.14 0.882 51.6 4.08 4.63 (2-(dimethylamino)ethanol) Pyridine C₅H₅N 79.1 0.979 36.7 0.88 0.92-Furanmethanol C₅H₆O₂ 98.1 1.127 53.3 4.62 4.1 (furfuryl alcohol)Methyl methacrylate C₅H₈O₂ 100.12 0.937 24.2 0.57 0.61N-Methyl-2-pyrrolidine C₅H₉NO 99.13 1.025 44.6 1.67 1.63 Isopropylacetate C₅H₁₀O₂ 102.13 0.871 22.3 0.52 0.6 (isopropyl ethanoate) Propylacetate C₅H₁₀O₂ 102.13 0.883 23.9 0.54 0.61 (propyl ethanoate)1,2-Dichlorobenzene C₆H₄Cl₂ 147 1.301 35.7 1.32 1.01 Benzene C₆H₆ 78.110.873 28.2 0.6 0.69 Aniline C₆H₇N 93.13 1.018 42.4 3.85 3.78Cyclohexanone C₆H₁₀O 98.14 0.942 34.4 2.02 2.14 Cyclohexane C₆H₁₂ 84.160.773 24.7 0.89 1.15 Cyclohexanol C₆H₁₂O 100.16 0.96 33.4 57.5 59.94-Methyl-2-pentanone C₆H₁₂O 100.16 0.796 23.5 0.55 0.69 (methyl isobutylketone) Butyl acetate C₆H₁₂O₂ 116.16 0.876 24.8 0.69 0.79 2-Methylpropyl C₆H₁₂O₂ 116.16 0.869 23 0.68 0.78 ethanoate (isobutyl acetate)2-Butoxyethanol C₆H₁₄O₂ 118.17 0.896 26.6 6.4 7.1 (ethylene glycolmonobutyl ether) Triethanolamine C₆H₁₅NO₃ 149.19 1.12 51.5 609 543Benzonitrile C₇H₅N 103.12 1.001 38.8 1.27 1.27 Benzaldehyde C₇H₆O 106.121.04 38.3 1.4 1.4 Toluene C₇H₈ 92.14 0.865 27.9 0.56 0.65 Benzyl alcoholC₇H₈0 108.14 1.041 36.8 5.47 5.25 3-Methylphenol C₇H₈O 108.14 1.03 35.812.9 12.5 (m-Cresol) 2-Heptanone (methyl C₇H₁₄O 114.19 0.811 26.1 0.710.88 n-amyl ketone) n-Heptane C₇H₁₆ 100.2 0.682 19.8 0.39 0.57 Styrene(phenylethene) C₈H₈ 104.15 0.9 32 0.7 0.78 Acetophenone C₈H₈O 120.151.024 39 1.68 1.64 (1-phenylethanone) Ethylbenzene C₈H₁₀ 106.17 0.86528.6 0.63 0.73 o-Xylene C₈H₁₀ 106.17 0.876 29.6 0.76 0.87 p-Xylene C₈H₁₀106.17 0.861 27.9 0.6 0.7 Phenoxyethanol C₈H₁₀O₂ 138.16 1.11 — 20.3 18.3(ethylene glycol monophenyl ether) Octanoic acid C₈H₁₆O₂ 144.21 0.90327.9 5.02 5.56 (caprylic acid) 2-Ethyl-1-hexanol C₈H₁₈O 130.23 0.83 27.76.27 7.55 Isophorone C₉H₁₄O 138.21 0.92 35.5 2.33 2.531-Bromonaphthalene C₁₀H₇Br 207.07 1.478 44.4 — — Dibutyl phthalateC₁₆H₂₂O₄ 278.34 1.043 37.4 16.6 15.9 Hexadecane C₁₆H₃₄ 226.44 0.77 27.1— — Dioctyl hexanedioate C₂₂H₄₂O₄ 370.57 0.92 — 13.7 14.9 (dioctyladipate) Bis(2- C₂₄H₃₈O₄ 390.56 0.98 31.1 80 82 ethylhexyl)phthalate(BEHP, dioctyl phthalate) Water H₂O 18.02 0.999 72.7 0.89 0.89 Hydrogenperoxide H₂O₂ 34.02 1.449 74 1.25 0.86 Hydrazine H₄N₂ 32.04 0.95 66.90.88 0.93 Mercury Hg 200.59 13.63 474.4 1.53 0.11 Silicon tetrachlorideSiCl₄ 169.9 1.645 18.8 99.4 60.4

Coatings solutions used to apply the coating configured to controlliquid flows in the microfluidic products of the invention can alsocomprise a liquid or mixture of liquids from the table above.

An embodiment of the invention is an analytical device wherein thegradient regions are created on a layer using the methods described inthe art cited in this application. The layer is then attached and/orbonded to one or more molded plastic layers containing a pattern forsample wells, analysis wells, and fluid passages wherein the fluidpassages comprise a top and bottom surface. The surfaces of the fluidpassages may be flat or non-flat. Dimensions for the widths of suchpassages can be in the range of 100-2500 microns, but the dimensions canbe smaller or larger depending on the manufacturing procedure used tocreate the fluid passages. In an embodiment, the wells are constructedto hold liquid volumes in the 0.2-200 microliter range, but can beconfigured to hold volumes down to the picoliter range. Wells andmanifolds can also be constructed to hold much higher volumes (1 mL ormore). In an embodiment, one or more of the fluid passages comprises acoating configured to control liquid flow wherein the coating comprisesa gradient surface energy coating from a proximal location on the fluidpassage to a distal location on a top or bottom surface of the fluidpassage. In an embodiment, the width of the coating is substantiallyequal to the width of the fluid passage. In another embodiment, thewidth of the coating can be greater or less than the width of the fluidpassages. In an embodiment, the top and bottom surfaces of the fluidpassages both comprise a coating configured to control liquid flow. Inanother embodiment, only one surface of the fluid passage comprises acoating configured to control liquid flow. In another embodiment, one ormore surfaces of the fluid passages comprise a substantially uniformcoating.

In embodiments, the microfluidic product comprises one or more fluidpassages wherein the width of the one or more fluid passages is nogreater than 3 millimeters. In embodiments, the microfluidic productcomprises one or more fluid passages wherein the width of the one ormore fluid passages is no greater than 2 millimeters. In embodiments,the microfluidic product comprises one or more fluid passages whereinthe width of the one or more fluid passages is no greater than 1millimeter. In embodiments, the microfluidic product comprises one ormore fluid passages wherein the width of the one or more fluid passagesis no greater than 0.8 millimeters. In embodiments, the microfluidicproduct comprises one or more fluid passages wherein the width of theone or more fluid passages is no greater than 0.5 millimeters. Inembodiments, the microfluidic product comprises one or more fluidpassages wherein the width of the one or more fluid passages is nogreater than 0.3 millimeters. In embodiments, the microfluidic productcomprises one or more fluid passages wherein the width of the one ormore fluid passages is no greater than 0.1 millimeters. In embodiments,the microfluidic product comprises one or more fluid passages whereinthe width of the one or more fluid passages is no greater than 0.05millimeters.

In embodiments, the microfluidic product comprises one or more fluidpassages wherein the width of the one or more fluid passages is no lessthan 0.05 millimeters. In embodiments, the microfluidic productcomprises one or more fluid passages wherein the width of the one ormore fluid passages is no less than 0.1 millimeters. In embodiments, themicrofluidic product comprises one or more fluid passages wherein thewidth of the one or more fluid passages is no less than 0.2 millimeters.In embodiments, the microfluidic product comprises one or more fluidpassages wherein the width of the one or more fluid passages is no lessthan 0.5 millimeters. In embodiments, the microfluidic product comprisesone or more fluid passages wherein the width of the one or more fluidpassages is no less than 0.7 millimeters. In embodiments, themicrofluidic product comprises one or more fluid passages wherein thewidth of the one or more fluid passages is no less than 1 millimeter. Inembodiments, the microfluidic product comprises one or more fluidpassages wherein the width of the one or more fluid passages is no lessthan 2 millimeters.

Specific embodiments that use different values for the height, widths,and length of the fluid passages are within the scope of this invention.Different polymers (polycarbonate, polyester, polypropylene, nylon,polyethylene, PTFE, PVDF, COC, etc.) can be used for the plastic layerscomprising the fluid passages. Different manufacturing techniques(machining, injection molding, additive manufacturing, etc) can be usedto manufacture the invention. Different attachment methods (overmolding,thermal welding, adhesives, gaskets, clamps, etc) can be used to contactor seal the layer with the gradient regions to the other layers of themicrofluidic product. In an embodiment, one or more registration markson a first component of the microfluidic product correspond to one ormore registration marks on a second component of the microfluidicproduct wherein the second component of the microfluidic productcomprises a coating configured to control liquid flows.

EXAMPLES Examples 1-4

Test slides such as those shown in FIG. 2A were made by laminating threelayers of material together to form a device with sample wells,channels, and exit wells.

In the test slides, the bottom layer 10 of the device was a gold-coatedMylar, 0.010″ nominal, 0.095″ measured polyester layer, the second layer20 with the well and channel patterns cut out of it was an 0.004″ thicklaminate composed of a 0.001″ thick layer of 3M 9461 Transfer Adhesivematerial on the top and bottom of a 0.002″ thick PET film, and the toplayer 30 was an Evonik CYRO LLC Acrylite FF 0.060″ Acrylic layer. Allchannels on the slides were cut to a width of 800 microns and a lengthof 1.5 cm. The top cover had lines 40 etched perpendicular to thechannels every 3 mm; these lines were used to determine the distancethat the liquid in the channel traveled over a given length of time.

To prepare surface energy gradient surfaces, the gold surfaces of thebottom layer were coated using thiol solutions to form self-assembledmonolayers on the gold. Hydrophobic coatings were formed by coating thegold surface with using 5-mM solutions of 1-dodecanethiol (availablefrom Aldrich Chemical Company) in 2-Propanol (available from VWRscientific). Hydrophilic coatings were formed using 5-mM solutions of11-Mercapto-1-undecanol in 2-Propanol. The contact angle formed with thehydrophobic coating was ˜116 degrees while the contact angle formed withthe hydrophilic coating was ˜25 degrees. Mixed solutions of varyingratios of the 1-dodecanethiol solution and the 11-mercapto-1-undecanolsolution were used to prepare coatings with contact angles between 116degrees and 20 degrees. No coating was applied to the sample well site.

To test the flow through each channel, a 2-microliter sample of water atroom temperature was added to the sample well. A Celestron DigitalMicroscope with video recording capabilities (Digital Microscope Suite2.0) was used to record the experiments. The video recording was used todetermine the time required for the water to travel each 3 mm incrementin the channels. Results of the experiments showed that liquid flowcould be controlled for any length of the channels tested (an aspectratio range of 0-18.75).

FIG. 1 is a graph for the necessary contact angle needed with the bottomsurface of the test slides to initiate capillary flow of water into thechannel using Equation 1. Based on the results of FIG. 1, nocapillary-driven flow would be expected with hydrophobic or bare goldsurfaces on the bottom layer for channels that were 800 microns wide by100 microns deep. The curves in FIG. 1 were generated for a channelwhere the top surface was a material that formed a contact angle of 60degrees with water. Using a different liquid besides water, or using adifferent material for the top surface would change the curves seen inFIG. 1.

Results showing how fluid velocities can be controlled by surface energygradients are shown in FIG. 3. As seen in the figure, no flow was seenfor the channels containing a hydrophobic coating or a bare goldsurface. For channels coated with the hydrophilic coating, it can beseen that the liquid took just under 3 minutes (180 seconds) to travel1.2 cm. The results show that gradients can be used to provide furthercontrol over the fluid velocity in the channel. The curves for Grad 1,Grad 2, and Grad 3 show that flow was initiated in the channel for allthree gradients, but the flow rates through the channel were differentfor each gradient. It is interesting to note that the flow rate for thegradient-coated channels appear to be more constant than the flow ratefor the hydrophilic-coated channels. In Grad1, the contact angle betweenwater and the bottom surface at the entrance of the channel was about100 degrees and the contact angle decreased at an average rate of about5 degrees/mm until it reached the end of the channel where the contactangel was about 25 degrees. In Grad2, the contact angle between waterand the bottom surface at the entrance of the channel was about 100degrees and the contact angle decreased at an average rate of about 2.5degrees/mm until it reached the end of the channel where the contactangle was about 60 degrees. In Grad3, the contact angle between waterand the bottom surface at the entrance of the channel was about 75degrees and the contact angle decreased at an average rate of about 3degrees/mm until it reached the end of the channel where the contactangle was about 25 degrees. The results show that both the compositionof the coating at the beginning of the channel as well as the degree ofthe surface energy gradient along the channel can be used to controlliquid flows.

FIG. 4 shows how surface energy gradients can be used to provide furthercontrol over liquid flows within a fluid passage. The results show thatgradient coatings can be used to stop, reduce, or accelerate the fluidflow within a channel. As seen in FIG. 4, no flow was seen in channelswith a bare-gold surface as expected. It can also be seen that theliquid flows do not reach the end of the channel for 2 of the surfaces(Lines 1 and 2). The liquid flow in the channel represented by Line 3did reach the end of the channel. Coatings for the channels used tocollect the data shown in Lines 1, 2, and 3 can be characterized asfollows:

-   -   Line 1: Contact angle with water at the channel entrance was        about 55 degrees and after 3 mm, the contact angle with water        changed to about 116 degrees.    -   Line 2: Contact angle with water at the channel entrance was        about 55 degrees and decreased by an average rate of 2.5        degrees/mm for about 6 mm and then the contact angle with water        changed to about 116 degrees for the remainder of the channel        length.    -   Line 3: Contact angle with water at the channel entrance was        about 55 degrees and decreased by an average rate of about 2.5        degrees/mm for about 9 mm to reach a contact angle of about 30        degrees and the contact angle stayed at about 30 degrees for the        remainder of the channel.

These results showed that the coatings can be configured in channelsthat are capable of stopping liquid flows (Line 1) and slowing andstopping liquid flows (Line 2). In addition, Line 3 shows that thecoatings of the invention can be used to increase liquid flow through achannel in comparison to uncoated surface and hydrophobically-coatedsurfaces. In the curve for Line 1, it can be seen that the liquid movesthrough the first 3 mm of the channel at an average velocity of about0.05 mm/s before stopping after 3 mm. For Line 2, the liquid flowedthrough the first 6-mm of the channel at an average velocity of about0.05 mm/s before the flow was decelerated and stopped; the averagevelocity for the liquid to move from the 6-mm point of the channel tothe 9-mm point of the channel was about 0.0125 mm/s and flow was stoppedafter 9-mm. For Line 3, the liquid flowed through the first 9-mm of thechannel at an average velocity of about 0.05 mm/s before it reached thepoint where the contact angle changed and the fluid flow wasaccelerated. Average fluid velocity for the last 3-mm of the channel wasabout 0.1 mm/s in Line 3.

Example 5

Glass slides were first coated with a solution of 1% octylsilane inethanol that contained sufficient acetic acid to lower the pH to 5.5.Slides were placed in solution for 5 minutes and then cured at 110 C inan oven for 2 hours.

Solutions Used for Coating

-   -   A) Solution A: Blend of 10 mg/ml PTFM (poly(2,2,2-trifluoroethyl        methacrylate)), 0.3 mg/ml PHFM (poly(hexafluorethyl        methacrylate)), and 0.5 mg/ml Isurlite® in methyl acetate    -   B) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer        MW 100,000) from BASF and 0.5 mg/ml Isurlite® in methyl acetate    -   C) Mixture of 30% Solution A and 70% Solution B    -   D) Mixture of 10% Solution A and 90% Solution B    -   E) Mixture of 5% Solution A and 95% Solution B    -   F) Mixture of 1% Solution A and 99% Solution B    -   G) Mixture of 0.1% Solution A and 99.9% Solution B    -   H) Mixture of 0.01% Solution A and 99.99% Solution B        Slides were then marked into 6 different regions and each region        was coated with solutions A-F shown above using the following        procedure:    -   1) The entire slide was immersed for 30 seconds in a 0.5 mg/ml        solution of Isurlite® in isopropyl alcohol. The slides were then        removed at a rate of ˜0.5 cm/sec.    -   2) Coated slides were air dried for ˜15 min    -   3) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.    -   4) For Slide 1, coating solutions were applied to specific        regions (˜0.4 square inches each) of each slide using a pipetter        and a 20-ul drop. Solutions were applied and spread on each        region to give an average liquid film thickness of ˜0.08 mm.    -   5) For Slide 2, coating solutions were applied to specific        regions (˜0.4 square inches each) of each slide using a pipetter        and a 10-ul drop. Solutions were applied and spread on each        region to give an average liquid film thickness of ˜0.04 mm.    -   6) Samples were allowed to dry at room temperature    -   7) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Results for the contact angle        measurements for the slides are shown in Table 1.

TABLE 1 Contact Angle Measurements for Coated Regions Coating Solution BF E D C A Slide 1 15 75 70 50 60 80 Coating Solution A D F G H B Slide 2105 52 45 36 35 15

Results indicated that the wet film thickness and drying time willaffect the final contact angles for mixtures. Even at lowconcentrations, the hydrophobic species in solution may stay in solutionwhile the solution dries, resulting in the hydrophobic species beingmore concentrated at the surface of the final coating. Thinner films andshorter drying times can limit the tendency of one species to becomeconcentrated at the surface of the coating.

Example 6

Glass slides were first coated with a solution of 1%octyltrimethoxysilane in ethanol that contained sufficient acetic acidto lower the pH to 5.5. Slides were placed in solution for 5 minutes andthen cured at 110 C in an oven for 2 hours.

Solutions Used for Coating

-   -   A) Solution A: Blend of 10 mg/ml PTFM, 0.3 mg/ml PHFM, and 0.5        mg/ml Isurlite® in methyl acetate    -   B) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer        MW 100,000) from BASF and 0.5 mg/ml Isurlite® in methyl acetate    -   C) Mixture of 10% Solution A and 90% Solution B    -   D) Mixture of 1% Solution A and 90% Solution B    -   E) Mixture of 0.1% Solution A and 99.9% Solution B    -   F) Mixture of 0.01% Solution A and 99.99% Solution B        Specific areas of the silanized slides were then further coated        with solutions A-F as shown in Table 2 using the following        procedure:    -   1) The entire slide was immersed for 30 seconds in a 0.5 mg/ml        solution of Isurlite® in isopropyl alcohol. The slides were then        removed at a rate of ˜0.5 cm/sec.    -   2) Coated slides were air dried for ˜15 min    -   3) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.    -   4) Coating solutions were applied to specific regions (˜0.8        square inches each) of each slide using a pipetter and a 20-ul        drop. (Slide 1 used 10-ul drops). Solutions were applied and        spread on each region to give an average liquid film thickness        of ˜0.04 mm.    -   5) Samples were allowed to dry at room temperature    -   6) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Results for the contact angle        measurements for the slides are shown in Table 3.

TABLE 2 Coating Patterns - Coating Solutions Used in Each Region ofSlides Coating Pattern Region 1 Region 2 Region 3 Region 4 1 A A A A 2 CD E F 3 D E F F 4 D D E F 5 F E D C

TABLE 3 Contact Angle Measurements for Each Coated Region Slide CoatingPattern Region1 Region 2 Region 3 Region 4 1 5 15 15 85 110 2 4 85 65 15<15 3 3 75 20 15 <15 4 2 105 85 15 15

Example 7

Specific portions of bare glass slides were coated with solutions A-Ffrom Example 6 using the following procedure:

-   -   1) The entire slide was immersed for 30 seconds in a solution of        Photoprime SR in isopropyl alcohol (either 10 mg/ml or 3 mg/ml).        The slides were then removed at a rate of ˜0.5 cm/sec.    -   2) Coated slides were air dried for ˜15 min    -   3) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.    -   4) Coating solutions were applied to specific regions (˜0.4        square inches each) of each slide using a pipetter and a 10-ul        drop. Solutions were applied and spread on each region to give        an average liquid film thickness of ˜0.04 mm.    -   5) Samples were allowed to dry at room temperature    -   6) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Results for the contact angle        measurements for the slides are shown in Table 4.

TABLE 4 Contact Angle Measurements for Coated Regions Coating Solution AC D E F B 10 mg/ml Photoprime SR ® 105 75 80 110 85 65 3 mg/mlPhotoprime SR ® 105 85 90 42 30 25

Results indicate that the higher concentration of PhotoprimeSR® resultedin coatings that retained a higher concentration of Photoprime SRO atthe surface. Coatings using a lower PhotoprimeSR® concentration showed ageneral decrease in contact angle as the concentration of thehydrophilic species increased.

Example 8

Specific portions of bare glass slides were coated with solutions A-Ffrom Example 6 as shown in Table 5 using the following procedure:

-   -   1) The entire slide was immersed for 30 seconds in a solution of        3 mg/ml Photoprime SR in isopropyl alcohol. The slides were then        removed at a rate of about 0.5 cm/sec. 2) Coated slides were air        dried for about 15 min    -   3) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of about 4 inches.    -   4) Coating solutions were applied to specific regions (about 0.4        square inches each) of each slide using a pipetter and a 10-ul        drop. Solutions were applied and spread on each region to give        an average liquid film thickness of about 0.04 mm.    -   5) Samples were allowed to dry at room temperature    -   6) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of about 4 inches.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Results for the contact angle        measurements for the slides are shown in Table 6.

TABLE 5 Coating Patterns - Coating Solutions Used in Each Region ofSlides Coating Pattern Region 1 Region 2 Region 3 Region 4 1 A A A A 2 CD E F 3 D E F F 4 D D E F 5 F E D C

TABLE 6 Contact Angle Measurements for Each Coated Region Slide CoatingPattern Region1 Region 2 Region 3 Region 4 1 3 65 30 24 15 2 4 62 65 2920 3 2 95 66 23 12 4 5 15 15 68 100

Example 9

Glass slides with a pattern as shown in FIG. 6 were coated withsolutions A-F from Example 6 as shown in Table 7. The slides were coatedaccording to the following procedure:

-   -   1) The entire slide was immersed for 30 seconds in a solution of        3 mg/ml Photoprime SRO in isopropyl alcohol. The slides were        then removed at a rate of about 0.5 cm/sec.    -   2) Coated slides were air dried for ˜15 min    -   3) The slides were then exposed to UV-C (254 nm peak) for 1 min        using UV-C at a distance of ˜4 inches.    -   4) Coating solutions were applied to specific region of each        slide using a pipette; volumes applied to each region are shown        in Table 7.    -   5) Samples were allowed to dry at room temperature    -   6) Samples were UV-cured for 5 minutes at a distance of 1-inch        using a Loctite Zeta 7735 UV-Curing system.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Results for the contact angle        measurements for the slides are shown in Table 8.

TABLE 7 Coating Patterns for Slides with Diamond Shape Region 1 2 3 4 56 7 8 9 x-axis dimension 7 7 4 10 2 2 4 4 10 mm mm mm mm mm mm mm mm mmDis- 1.1 1.1 1.6 6.4 0.8 0.8 0.6 0.6 2.4 pense Volume (micro- litersSlide 1 B B B B B B B B B Slide 2 D D D D E F B B B Slide 3 C D D D E FB B B Slide 4 C D D D E F B C A

TABLE 8 Contact Angle for Each Coated Region Region 1 2 3 4 5 6 7 8 9x-axis dimension 7 7 4 10 2 2 4 4 10 Slide mm mm mm mm mm mm mm mm mm 1<15 <15 <15 <15 <15 <15 <15 <15 <15 2 55 55 45 40 25 20 20 20 15 3 60 4548 45 30 30 20 15 15 4 60 50 50 45 30 25 25 60 90

Example 10

Experiments were then performed to produce coatings on polyester. A20-mm wide strip of Duralar® polyester (0.005″ thick) was cleaned withisopropyl alcohol, eleven 16-mm long sections were marked on the slide,and each section was then coated with the solutions shown in Table 9using the following procedure:

-   -   1) 10-ul volumes were dispensed over each region and spread over        each region with a pippetter; the average liquid film thickness        was about 0.03 mm.    -   2) Coatings were allowed to dry at room temperature.    -   3) Samples were UV-cured for 5 minutes at a distance of 1-inch        using a Loctite Zeta 7735 UV-Curing system.        Afterwards, contact angle measurements were taken using 0.5-uL        drops of deionized water. Three measurements were taken for each        section. Results for the contact angle measurements for the        samples are shown in Table 9.

TABLE 9 Contact Angle Measurements for Coatings on Polyester ContactAngle Section Coating Solution after UV-cure 1 None (no UV-cure) 75 2Solution B from Ex. 6 20 3 20 mg/ml poly(2,2,2-trifluoromethacrylate)115 in methyl acetate 4 Solution A from Ex. 6 120 5 0.5 mg/ml Isurlite ®in isopropyl alcohol 58 6 None 60 7 5 mg/mL benzophenone in isopropylalcohol 58 8 5 mg/ml 4,4′-dihodroxy benzophenone in 48 isopropyl alcohol9 5 mg/ml 4,4′-difluoro-benzophenone in 105 isopropyl alcohol 10Solution D from Ex. 6 45 11 Solution E from Ex. 6 30

Example 11

Polyester strips were coated with solutions using 2 differentconcentrations of Isurlite®; 0.5 mg/mL and 2.0 mg/mL. A 20-mm wide stripof Duralar® polyester (0.005″ thick) was cleaned with isopropyl alcohol,twelve 16-mm long sections were marked on the slide, and each sectionwas then coated with the solutions shown in Table 10 and Table 11 usingthe following procedure:

-   -   1) 10-ul volumes were dispensed over each region and spread over        each region with a pippetter; the average liquid film thickness        was about 0.03 mm.    -   2) Coatings were allowed to dry at room temperature.    -   3) One set of samples were then exposed to UV-C (254 nm peak)        for 1 min using UV-C at a distance of about 4 inches.    -   4) One set of samples were then exposed to UV-C (254 nm peak)        for 2 min using UV-C at a distance of about 4 inches.        Afterwards, samples were rinsed with deionized water and allowed        to dry at room temperature. Contact angle measurements were then        taken using 0.5-uL drops of deionized water. Three measurements        were taken for each section. Results for the contact angle        measurements for the samples are shown in Table 9.

TABLE 10 Contact Angle Measurements for 1-min UV Cure Contact AngleSection Coating Solution after 1-min UV-cure 1 A 118 2 C 60 3 D 55 4 E42 5 F 22 6 B 39 7 None 60 8 H 72 9 I 53 10 J 30 11 K 21 12 G 16

TABLE 11 Contact Angle Measurements for 2-min UV Cure Contact AngleSection Coating Solution after 2-min UV-cure 1 A 90 2 C 53 3 D 45 4 E 405 F 35 6 B 27 7 None 62 8 H 75 9 I 53 10 J 30 11 K 24 12 G 25

-   -   A) Solution A: Blend of 10 mg/ml PTFM, 0.3 mg/ml PHFM, and 0.5        mg/ml Isurlite® in methyl acetate    -   B) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer        MW 100,000) from BASF and 0.5 mg/ml Isurlite® in methyl acetate    -   C) Mixture of 10% Solution A and 90% Solution B    -   D) Mixture of 1% Solution A and 90% Solution B    -   E) Mixture of 0.1% Solution A and 99.9% Solution B    -   F) Mixture of 0.01% Solution A and 99.99% Solution B    -   G) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer        MW 100,000) from BASF and 2.0 mg/ml Isurlite® in methyl acetate    -   H) Mixture of 10% Solution A and 90% Solution G    -   I) Mixture of 1% Solution A and 90% Solution G    -   J) Mixture of 0.1% Solution A and 99.9% Solution G    -   K) Mixture of 0.01% Solution A and 99.99% Solution G

Example 12

Coatings were applied for on a substrate in a pattern similar to the oneshown in FIG. 7. Dimensions for an embodiment used in this example areshown in FIG. 7. The substrate was Duralar® polyester (0.005″ thick).Each straight channel in the part was coated with a differentcomposition. The inlet channel region and manifold sections were alsocoated with different coating compositions. Coatings were dispensed ontoeach region to give a wet film thickness of 0.03-0.04 mm. Dispensevolumes varied from 100 nL-1 uL for each region depending on the area ofthe region. After the solutions were dispensed onto each region, theywere allowed to dry at room temperature. The coated materials were thenUV-cured for 1 minute using UV-C(254 nm peak) at a distance of about 4inches.

Coatings were applied to create a gradient coating from the inlet port700 to the expansion region 701 and manifold region 702 and through eachof the three straight channel sections 710, 720, and 730. For thestraight channel sections, coatings were applied along 4 equally-spaced5-mm lengths A, B, C, and D of each channel. In addition, a coating wasapplied to the entrance region 740A of the waste channel 740 in order toprevent/delay flow of liquid into the waste channel until the liquidfilled at least one straight channel sections.

The following coating solutions were used in this example:A) Solution A: Blend of 10 mg/ml PTFM, 0.3 mg/ml PHFM, and 0.5 mg/mlIsurlite® in methyl acetateB) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer MW100,000) from BASF and 2.0 mg/ml Isurlite® in methyl acetate

C) Mixture of 10% Solution A and 90% Solution B D) Mixture of 5%Solution A and 95% Solution B E) Mixture of 1% Solution A and 90%Solution B F) Mixture of 0.1% Solution A and 99.9% Solution B G) Mixtureof 0.01% Solution A and 99.99% Solution B

Four separate coating patterns were created by dispensing solutions A-Gonto specific regions of the substrate as shown below in Table 12.

TABLE 12 Coating Patterns for Multi-Channel Example Coating SolutionsUsed for Patterns in Multi-Channel Part Coated Area Dimensions Max CoatCoat Coat Coat Length Width Area 1 Pat- 2 Pat- 3 Pat- 4 Pat- Region (mm)(mm) (mm2) tern tern tern tern 701   3 2 4.5 C C C None 702   9 8 52.5 DD D None 710A 5 1 5 F G E G 710B 5 1 5 G G F G 710C 5 1 5 B B G B 710D 51 5 B B B B 720A 5 1 5 E E F C 720B 5 1 5 E E G D 720C 5 1 5 F E B D720D 5 1 5 B B B D 730A 5 1 5 F F E C 730B 5 1 5 F F E C 730C 5 1 5 F FF D 730D 5 1 5 G B G D 740A 4 0.8 3.2 A A A A 740   34 0.8 27.2 NoneNone None NoneContact angle measurements were then taken for each coating pattern aswell as for uncoated materials. 200-nL drops of deionized water wereused for the contact angle measurements. Results for the contact anglemeasurements are shown in Table 13.

TABLE 13 Contact Angle Measurements for Multi-channel Example ContactAngles for Regions in each Coating Pattern Region Uncoated Coat 1 Coat 2Coat 3 Coat 4 701   60 72 64 67 60 702   55 52 48 49 60 710A 59 33 21 3925 710B 59 22 15 30 25 710C 60 15 20 22 15 710D 60 20 20 14 10 720A 6048 45 27 70 720B 60 41 30 24 60 720C 56 30 30 11 60 720D 58 15 15 11 60730A 59 33 23 42 70 730B 59 32 22 39 70 730C 66 33 24 24 60 730D 55 2115 18 60 740A 59 115 114 105 110 740   63 60 65 60 60

Results of the experiment showed that gradients were created along thelength of the part from the inlet port through each channel. Resultsalso showed that different gradients were created with varying degreesof gradient in the channel sections.

A microfluidic part was then assembled using the substrate coated withcoating pattern 4 as the bottom layer, the adhesive layer in Example 1as the second layer, and uncoated 0.005″ thick Duralar® polyester as thetop layer. 10-ul, 6-ul, and 6-ul volumes of water were dispensed to theinlet of the port by a pipette tip using gravity flow. After 10-ul wasdispensed, all the fluid bypassed side channel 740 due to thehydrophobic coating in 740 A. All the initial liquid was directed intochannel 710 and did not enter channels 720 or 730. After subsequentadditions of the 6-ul water volumes, the liquid flowed into channels 720and 730 but only after channel 710 was filled. The flow rates in allthree channels was different due to the difference the gradient coatingsin each channel. Total time to flow through channel 710 was about 32seconds; total time to flow through channel 720 was about 47 seconds;and total time to flow through channel 730 was about 67 seconds. Oncethe three channels 710, 720, and 730 were filled, the pressure in theproduct was high enough for liquid to then flow into channel 740. Theseresults show that fluid flow can be controlled in channels in fluidcommunication with each other using gradient coatings. The coatings canbe used to control the order of channel entry and fluid velocities formultiple channels in fluid communication with each other.

Example 13

Gradient coatings were produced on plastic microfluidic productscomprising 6 individual channels that were each 1-mm wide, 100 micronshigh, and 15-mm in length. Coatings were applied to a 0.010″ thickacrylic which was then used as the bottom layer of the product. Theadhesive channel layer made of the same material as in Example 1 wasused as the 2^(nd) layer, and an uncoated 0.010″ thick acrylic was usedas the top layer.

The following coating solutions were used in this example:A) Solution A: Blend of 10 mg/ml PTFM, 0.3 mg/ml PHFM, and 0.5 mg/mlIsurlite® in methyl acetateB) Solution B: 10 mg/ml of Pluronic F38 (PEG/PPO block-copolymer MW100,000) from BASF and 2.0 mg/ml Isurlite® in methyl acetate

C) Mixture of 10% Solution A and 90% Solution B D) Mixture of 5%Solution A and 95% Solution B E) Mixture of 1% Solution A and 99%Solution B F) Mixture of 0.1% Solution A and 99.9% Solution B G) Mixtureof 0.01% Solution A and 99.99% Solution B

Coatings were applied in every 3-mm length section of the channels asshown in Table 13:

TABLE 13 Coating Pattern For Channel Regions Coating pattern Channel 0-3mm 3-6 mm 6-9 mm 9-12 mm 12-15 mm 1 None None None None None 2 A A A A A3 A A A D D 4 A D E E E 5 C F G G G 6 D F B B B2.2 ul of 60 dyne/cm Accudyne Test fluid (Diversified Enterprises,Claremont, N.H.) were then delivered to the inlet port of each channelat the same time using a multi-tip pipettor. The times required to flowacross each region of the channel were recorded and are shown in Table14.

Results show that a wide range of flow rates can be achieved with theproducts of the invention. In this example, flow rates from 0.014 mm/sup to 3 mm/s were achieved, all by changing the coating over eachchannel region. In addition, flow could be accelerated and deceleratedby changing the coating in each channel region.

TABLE 14 Flow times for Liquid in Each Channel Region Time (seconds)Required to Travel Across Channel Region Channel 0-3 mm 3-6 mm 6-9 mm9-12 mm 12-15 mm 1 3 3 4 1 2 2 30 32 83 150 215 3 30 65 35 24 18 4 30 1545 13 8 5 25 3 27 8 11 6 11 22 4 4 3

Products of the invention can be single or multi-layered cartridges,disks, or other component and can have a rectangular, circular, oval,racetrack, or any other shape. Products can be manufactured from any ofthe materials described previously. Products of the invention can haveadditional product design features that are useful in the transport andanalysis of liquids in microfluidic products.

In an embodiment, a microfluidic product according to the inventioncomprises at least one substrate. A substrate may include one or moreexpansions or areas along a channel or fluid passage. The substrates ofthe product may comprise an array of connected fluid passages. In oneembodiment, a microfluidic product is provided that includes one or moresubstrates comprising a first channel comprising an inlet separated froman outlet and one or more secondary channels (or branch channels) influid communication with the first channel.

The microfluidic product may comprise one or more outlet ports or inletports. Each of the outlet and inlet ports may also communicate with awell or reservoir. The inlet and outlet ports may be in fluidcommunication with the channels or reservoirs that they are connectingor may contain one or more valves. Fluid can be introduced into thechannels via the inlet by any means. In an embodiment, the inlet portsof the microfluidic product have a shape like that shown in FIG. 5. Thisinlet port design is useful for encouraging liquid to contact theentrance of a channel in microfluidic products. In an embodiment, theport can be designs by designing a semicircle 0.016″ smaller in diameterthan 3 times the desired channel width. The semicircle can then becentered one radius away from the channel entrance center point. Theremaining half of the inlet port design is an elliptical arc with majoraxis length 2 times the minor axis length, centered at the semicirclecenter. In another embodiment, the inlet port opening is smaller on thetop layer of the microfluidic product than it is on the bottom layer ofthe microfluidic product. The sample inlet may intercept the firstchannel at any angle. A first channel may in turn communicate with twoor more branch channels at another junction or “branch point”, forming,for example, a T-shape or a Y-shape. Other shapes and channel geometriesmay be used as desired.

In one embodiment of the invention, a microfluidic product comprises atleast one inlet port in fluid communication with a first channel, adetection region in fluid communication with the first channel, and adetector associated with the detection region. In an embodimentaccording to the invention, the microfluidic product comprises adetection region along a channel. There may be a plurality of detectionregions and detectors, working independently or together, e.g., toanalyze one or more properties of a chemical such as a reagent.

In an embodiment, a microfluidic product can be fabricated with a fluidreservoir or well at the inlet port, which is typically in fluidcommunication with an inlet channel. A reservoir preferably facilitatesintroduction of fluids into the substrate and into the first channel. Aninlet port may have an opening such as in the floor of the substrate topermit entry of the sample into the device. The inlet port may alsocontain a connector adapted to receive a suitable piece of tubing, suchas Teflon® tubing, liquid chromatography or HPLC tubing, through which afluid may be supplied

In an embodiment, the fabricated fluid passages and other components arecovered and sealed, often with a transparent cover, although other clearor opaque cover materials may be used. Analytical devices havingchannels, valves, and other elements can be designed and fabricated fromvarious materials. A variety of channels for sample flow and mixing canbe fabricated and can be positioned at any location on the product asthe detection and discrimination or sorting points. In embodiments,channels can also be designed into the microfluidic product that placethe fluid flow at different times/distances into a field of view of adetector. Channels can also be designed to merge or split fluid flows atprecise times/distances.

In an embodiment, a group of manifolds (a region consisting of severalfluid passages that lead to or from a common fluid passage) can beincluded to facilitate the movement of liquid through the microfluidicproduct. The outlet can be adapted for receiving, for example, a segmentof tubing or a sample tube.

In an embodiment, the microfluidic products of the invention can includeassay modules, preferably assay cartridges. An assay module of theinvention incorporates one or more fluidic components such ascompartments, wells, chambers, fluidic conduits, fluid ports/vents,valves, and the like and/or one or more detection components such aselectrodes, electrode contacts, sensors (e.g. electrochemical sensors,fluid sensors, mass sensors, optical sensors, capacitive sensors,impedance sensors, optical waveguides, etc.), detection windows (e.g.windows configured to allow optical measurements on samples in thecartridge such as measurements of absorbance, light scattering, lightrefraction, light reflection, fluorescence, phosphorescence,chemiluminescence, electrochemiluminescence, etc.), and the like. Amodule may also comprise reagents for carrying out an assay such asbinding reagents, detectable labels, sample processing reagents, washsolutions, buffers, etc. The reagents may be present in liquid form,solid form and/or immobilized on the surface of solid phase supportspresent in the cartridge. In certain embodiments of the invention, themodules include all the components necessary for carrying out an assay.In other embodiments, the invention also includes a module readeradapted to receive the module and carry out certain operations on themodule such as fluid movement, supplying power, conducting physicalmeasurements on the cartridge, and the like.

In another embodiment, the microfluidic product comprises a detectionregion having one or more binding domains having immobilized bindingreagents wherein the detection region is in fluid communication with thesample inlet. In an embodiment, the detection region comprises one ormore immobilized binding reagents and comprises a signal indicator whenspecies are bound to the binding reagents. The signal may be anelectrochemiluminescent signal wherein the detection chamber furthercomprises electrodes. The one or more binding reagents can comprise oneor more electrochemiluminescent labels.

In an embodiment, one or more fluidic networks may be defined within thecartridge's body by one or more cover layers mated to a side of thecartridge body. A second cover layer, or set of cover layers, may bemated to a second side of the cartridge body to form one or moreadditional second side fluidic networks therebetween, the first andsecond side fluidic networks being in fluidic communication by at leastone though-hole within the cartridge body. The fluidic networks may bedefined, at least in part, by recesses in the cartridge body and/orcover layers. In addition, at least one of the fluidic networks may bedefined, at least in part, by apertures or openings within a gasketlayer disposed between the cartridge body and at least one cover layer.

In another embodiment, the microfluidic product may comprise air ventports.

In an embodiment, the microfluidic products of the invention comprise aplurality of flow cells or detection chambers. In certain embodimentsthe flow cell may comprise the same assay domains or, at least, have atleast some assay domains that share specificity for the same analytes ofinterest. In these embodiments, the plurality of flow cells may be usedto analyze a plurality of different samples or to compare samples thathave been pre-treated in different ways. Alternatively, one of the flowcells may be a control flow cell used to analyze a control sample andanother of the flow cells may be a test flow cell used to analyze a testsample. The control sample may be a completely pre-defined controlsample or may be a mixture comprising the test sample but spiked withadded analytes of interest so as to allow for calibration of the assaysby the method of standard addition. In an alternative embodiment, themicrofluidic product has at least two flow cells that have assay domainsfor two different assay panels. Advantageously, such a product may beused to separately perform assay reactions that are incompatible witheach other.

In a preferred embodiment of the invention, the microfluidic product hasminimal or no active mechanical or electronic components.

In other embodiments, the microfluidic product is introduced onto acartridge reader to carry out an assay. For example, a reader may haveelectronic circuitry for applying electrical energy to the assayelectrodes and for measuring the resulting potentials or currents atassay electrodes. The reader may have one or more light detectors formeasuring luminescence generated at assay electrodes. Light detectorsthat may be used include, but are not limited to photomultiplier tubes,avalanche photodiodes, photodiodes, photodiode arrays, CCD chips, CMOSchips, film. The light detector may be comprised within an opticaldetection system that also comprise lenses, filters, shutters,apertures, fiber optics, light guides, etc. The reader may also havepumps, valves, heaters, sensors, etc. for providing fluids to thecartridge, verifying the presence of fluids and/or maintaining thefluids at an appropriate controlled temperature. The reader may be usedto store and provide assay reagents, either onboard the reader itself orfrom separate assay reagent bottles or an assay reagent storage device.The reader may also have cartridge handling systems such as motioncontrollers for moving the cartridge in and out of the reader. Thereader may have a microprocessor for controlling the mechanical and/orelectronic subsystems, analyzing the acquired data and/or providing agraphical user interface (GUI). The cartridge reader may also compriseelectrical, mechanical and/or optical connectors for connecting to thecartridge.

In an embodiment, the components of the microfluidic products can bedesigned and incorporated into the cartridge to form the fluidic networkusing certain predefined design guidelines. The design guidelines foreach component can be dependent upon one or more factors such as, e.g.,cartridge body design (i.e., single-piece body, multiple piece body,modular body, single read chamber, multiple read chamber, and the like),manufacturing process (e.g., injection molding, blow molding, hotstamping, casting, machining, etc.), materials (e.g., acrylic, PVDF,PET, polystyrene, polypropylene and the like), assay requirements (e.g.,binding assay, competitive binding assay, single step assay, two-stepassay, etc.), functional requirements (e.g., sample size, assay reagentvolumes, detection technology, time-to-result, incubation, heating,mixing/agitating), safety/handling requirements (e.g., self-containment,regulatory approval, ease of use, etc.), and/or the like.

The skilled practioner will be able to readily select materials suitablefor the fabrication of the microfluidic products of the invention.Suitable materials include glass, ceramics, metals and/or plastics suchas acrylic polymers (such as Lucite), acetal resins (such as Delrin),polyvinylidene fluoride (PVDF), polyethylene terephthalate (PET),polytetrafluoroethylene (e.g., Teflon), polystyrene, polypropylene, ABS,PEEK and the like. Preferably, the materials are inert to anysolutions/reagents that will contact them during use or storage of themicrofluidic product. In certain embodiments, at least some portion ofthe microfluidic product is fabricated from transparent and/ortranslucent materials such as glass or acrylic polymer to providewindows that allow optical interrogation of fluids or surfaces insidethe cartridge, e.g., for analysis of compositions within detectionchambers of the cartridge or for monitoring and controlling the movementof liquids through the fluidic networks defined within the cartridge.

An embodiment of the microfluidic product is a cartridge comprising acoating configured to control liquid flow wherein the cartridge includesone or more sample chambers, one or more detection chambers (preferably,detection chambers adapted for use in ECL measurements as describedabove) and one or more waste chambers. The chambers are connected in byfluid passage so that a sample introduced into a sample chamber can bedelivered into one or more detection chambers for analysis and thenpassed into one or more waste chambers for disposal. This cartridge mayinclude one or more reagent chambers for storing liquid reagents, thereagent chambers connected via fluid passages to the other components soas to allow the introduction of the liquid reagents into specifiedsample or detection chambers. The cartridge may also include vent portsin fluidic communication with the sample, detection and/or wastechambers (directly or through vent conduits) so as to allow theequilibration of fluid in the chambers with the atmosphere or to allowfor the directed movement of fluid into or out of a specified chamber.

In an embodiment, the microfluidic cartridge comprises a sample chamberwherein the sample chamber is a chamber defined within a cartridge thatis adapted for receiving a sample to be analyzed in the cartridge. Thesample chamber includes a sample introduction port for introducingsample into the chamber. The port is preferably an opening in thecartridge that provides access to the sample chamber. Alternatively, theport may be a membrane or septa through which a sample may be injectedinto the sample chamber, e.g., through the use of a needle or cannula.In an embodiment, the cartridge also includes a sealable closure forsealing the sample introduction port and preventing leakage of thesample and possible exposure of the user and/or associated instrumentsto biohazards. Use of a modular detachable insert within the samplechamber also allows for independent selection of materials for the maincartridge body. In an alternative embodiment, sealing of the sampleintroduction port is achieved by applying an adhesive tape to the port.The sample chamber may contain dry reagents used in carrying out theassay that reconstitute on addition of a liquid sample. Optionally, thesample chamber contains an anti-foam agent to prevent foaming of thesample in the cartridge. In an embodiment, the sample chamber comprisesthe coating configured to control liquid flow in order to prevent leaks.

In one embodiment, the microfluidic product comprises a sample chamberwherein the sample chamber comprises a sample introduction port whereinthe sample introduction port aperture also acts as a vent port. The ventport may also be provided through the top of the sealing/cappingmechanism by, e.g., incorporating a vent hole in the top surface of thesealing/capping mechanism. An alternative embodiment may employ a schemewhereby the cartridge reader itself can include a piercing/ventingmechanism that is adapted and configured to pierce through the topsurface of the flexible sealing/capping mechanism. In an embodiment, thesealing/capping mechanism is adapted and configured to be self-sealingupon withdrawal/removal of the piercing/venting mechanism, e.g., via theuse of a septum preferably comprising an elastomeric material. In anembodiment, the sample chamber comprises the coating configured tocontrol liquid flow in order to prevent leaks.

In an embodiment, the sample chamber may also include a filter for,e.g., removing particulate matter that may be present within the sampleitself or that may be present as a result of using a swab or the like tointroduce sample into the sample chamber. An embodiment may employ afilter that not only removes any particulate matter but that is alsodesigned to separate red blood cells (RBC) from blood plasma; e.g.,where the particular assay/assay format requires blood plasma as thesample. Such a filter can be an integral cross-flow filter, in-linefilter or the like. In an embodiment, the filter is arranged at or nearthe entrance of the sample conduit.

In an embodiment, microfluidic products may also comprise a cartridgethat comprises a reagent module. The reagent module can comprise acontainer such as an ampoule (e.g., glass, plastic, or the like), apouch (e.g., plastic, metal foil, plastic/metal foil laminates, rubber,or the like), a blister pack, a syringe, or the like, or any othercontainer that can be filled with fluid, sealed and dropped into thecartridge for subsequent fluid delivery. Preferred materials includeglass, plastics with good water vapor barrier properties (e.g., cyclicolefin copolymers such as copolymers of ethylene and norbornene, nylon6, polyethyelene naphthalate, polyvinylidene chloride andpolychlorotrifluoroethylene) and metal foil/plastic laminates because oftheir chemical inertness and their resistance to evaporative losses,other suitable materials will be apparent to the skilled practitioner.Ampoules can comprise a material that can be made to shatter or break onimpact such as glass or hard plastic. Embodiments incorporatingbreakable ampoules preferably also include filters to ensure thatsubstantially all of the fragments that may result upon rupturing theampoules are not permitted to enter the fluidic network and possiblyobstruct/block fluid flow.

In an alternative embodiment, a pierceable container such as a pouch orblister pack may be employed. Preferably, the pierceable container has apierceable wall made from a plastic film, a metal foil, or mostpreferably, a metal foil/plastic film laminate. In such an embodimentthe assay reagent release mechanism could employ a piercing scheme. Inanother alternate embodiment, liquid reagents are stored in a syringecomprising a syringe chamber and a plunger. The chamber may be anintegral component of the cartridge, a module that is inserted into thecartridge or a separate component that is attached (e.g., via a luerlock connection) to the cartridge prior to use. Actuation of the plungermay be used to release the contents of the syringe into a reagentchamber or, alternately, to transfer the contents directly into otherfluidic components of the cartridge.

In an embodiment, the microfluidic product can comprise waste chamberslinked to a waste chamber conduit and, preferably, to a vent port (e.g.,through a vent conduit). The waste chamber can be configured to allowliquid waste to be delivered to the waste chamber through the wastechamber conduit and, preferably, for air that is included in the wastestream to escape through a waste chamber vent port. Optionally, thewaste chambers contain a water absorbing material, such as a sponge,that retains waste fluid and prevents leakage of the waste fluid ondisposal of a cartridge.

In an embodiment, the microfluidic product comprises a cartridgecomprising detection chambers. The detection chambers are adapted forcarrying out a physical measurement on the sample. If the measurementrequires illumination or optical observation of the sample (e.g., as inmeasurements of light absorbance, photoluminescence, reflectance,chemiluminescence, electrochemiluminescence, light scattering and thelike) the detection chamber should have at least one transparent wallarranged so as to allow the illumination and/or observation. Whenemployed in solid phase binding assays, the detection chamber preferablycomprises a surface (preferably, a wall of the chamber) that has one ormore binding reagents (e.g., antibodies, proteins, receptors, ligands,haptens, nucleic acids, etc.) immobilized thereon. In an embodiment, thedetection chamber is an electrochemiluminescence detection chamberhaving one or binding reagents immobilized on one or more electrodes. Inone embodiment, the cartridge comprises a working electrode having anarray of binding reagents immobilized thereon. In another embodiment,the cartridge comprises an array of independently controllable workingelectrodes each having a binding reagent immobilized thereon.

A cartridge may comprise one or more detection chambers. Cartridgescomprising multiple detection chambers may comprise separate fluidicsystems for each detection chamber (e.g., multiple sample chambersand/or reagent chambers and associated fluidic conduits) so that assayson multiple samples may be carried out in parallel. In embodiments,multiple detection chambers are linked to a single sample chamber andmay share the use of other fluidic components such as reagent chambers,waste chambers and the like. In these embodiments, the two detectionchambers may be used to carry out different sets of assays, thusincreasing the number of measurements that can be carried out on asample relative to a cartridge with one detection chamber.Advantageously, the use of multiple detection chambers allows forcarrying out in a single cartridge multiple incompatible measurements,that is measurements that cannot be performed in a single reactionvolume or benefit from being carried out in separate reaction volumes,e.g., measurements that have different requirements for pH or assaycomposition or otherwise negatively interfere with each other.

In another embodiment the microfluidic product comprises a plurality ofdetection chambers wherein one or more of a plurality of detectionchambers is used as control/calibration chamber for measuring assaycontrol/calibration samples. In one such embodiment, a first and asecond detection chamber are each configured to carry out a panel of oneor more assays for one or more analytes. One detection chamber (the testchamber) is used to analyze a sample. The other detection chamber (thecontrol chamber) is used to analyze a spiked sample having apredetermined additional amount of the one or more of the analytes ofinterest. The change in signal between the two chambers allows for thecalculation of the responsivity of the signal to changes in analyte andcan be used to calibrate the system and/or to determine if the cartridgeis functioning properly. In another embodiment employing a controlchamber, the control chamber is not used to analyze the sample or aderivative thereof but is used to measure analyte in a separate controlor calibrator matrix. The signal in the control chamber may be used fordetermining background signals (by using a matrix with no analyte), forcalibrating the instrument (by using a calibrator matrix with apredetermined amount of analyte to determine calibration parameters) orto determine if the cartridge is functioning properly (by using acontrol matrix with a predetermined amount of analyte and determining ifthe signal falls within a predetermined acceptable range).

In another embodiment of the microfluidic product, the cartridgefluidics may include bubble traps. The bubble trap is a chamber orconduit adapted for removing bubbles from fluid streams. In anembodiment, there is a bubble trap between the sample and detectionchambers so that bubbles in the sample may be removed prior tointroducing the sample into the detection chamber.

Although some embodiments of the microfluidic products of the inventioncan operate without valves to control fluid flow under different flowconditions, in some embodiments, the microfluidic products may alsocomprise valves to provide additional control the flow of fluid throughthe cartridge. A variety of suitable valves (including mechanicalvalves, valves based on electrokinetic flow, valves based ondifferential heating, etc.) will be known to one of average skill in theart of assay cartridges or microfluidic devices. In one embodiment, afluid conduit has a flexible wall/diaphragm that in the absence ofexternal force allows fluid to pass through the conduit. Application ofan external force on the wall/diaphragm (e.g., from a piston or via theapplication of gas or hydrostatic pressure) causes the diaphragm toimpinge on the conduit, thus impeding the flow of fluid.

In an embodiment, the fluidic network of the microfluidic products mayinclude at least one viscosity measuring conduit, preferably linked to asample chamber or sample conduit, having an inlet and an outlet. Theconduit is adapted so that a liquid sample can be introduced into theconduit and the time it takes the liquid to move between two locationsin the conduit can be timed. Such an arrangement can advantageously beused to measure clotting times of a blood or plasma sample, or fordetermining viscosity changes of a liquid under different flow andtemperature conditions.

In an embodiment, the microfluidic product comprises vent ports withapertures on the surface of the cartridge that are in fluidiccommunication with fluidic chambers or fluid passages within thecartridge. In an embodiment, the microfluidic product comprises alaminated cartridge construction comprising vent ports that are providedby apertures in cover layers that seal against a cartridge body todefine planar fluidic networks or alternatively, by through-holesexposed on one surface of the cartridge body that communicate withfluidic networks on the opposing side. In an embodiment, the vent portsare used to introduce air into liquid streams passing through thefluidic conduits of the invention, for example, to segment the fluidstreams with slugs of air. The introduction of air may be used toprevent mixing of two liquid slugs passed sequentially through aconduit, to clear a liquid from a conduit and/or to enhance theefficiency of a wash step. In an embodiment, the vent ports are arrangedin a single row at a common location along the cartridge body's width.

Assays Utilizing Microfluidic Products of the Invention

In an embodiment, the microfluidic product of the invention comprisesone or more assay cartridges. The assay cartridges may be used to carryout panels of assays. Suitable panels include panels of assays foranalytes or activities associated with a specific biochemical system,biochemical pathway, tissue, organism, cell type, organelle, diseasestate, class of receptors, class of enzymes, class of pathogen,environmental sample, food sample, etc. Preferred panels includeimmunoassay for cytokines and/or their receptors, growth factors and/ortheir receptors, second messengers (e.g., cAMP, cGMP, phosphorylatedforms of inositol and phosphatidyl inositol, etc.) drugs of abuse,therapeutic drugs, auto-antibodies (e.g., one or more antibodiesdirected against the Sm, RNP, SS-A, SS-B Jo-1, and Scl-70 antigens),allergen specific antibodies, tumor markers, cardiac markers (e.g., oneor more of Troponin T, Troponin I, myoglobin, CKMB, etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (beta-amyloid,tau-protein, etc.), markers of osteoporosis (e.g., one or more ofcross-linked N or C-telopeptides, total deoxypyridinoline, freedeoxypyridinoline, osteocalcin, alkaline phosphatase, C-terminalpropeptide of type I collagen, bone-specific alkaline phosphatase,etc.), markers of fertility (e.g., one or more of Estradiol,progesterone, follicle stimulating hormone (FSH), luetenizing hormone(LH), prolactin, beta-hCG, testosterone, etc.), markers of congestiveheart failure, markers of thyroid disorders, and markers of prostratecancer (e.g., one or more of total PSA, free PSA, complexed PSA,prostatic acid phosphatase, creatine kinase, etc.), pathogens associatedwith upper respiratory infection (e.g., influenza A, influenza B,Respiratory Syncytial Virus, Streptococci species), pathogens found infood and water (e.g., salmonella, listeria, cryptosporidia,campylobacter, E. Coli 0157, etc.), sexually transmitted diseases (e.g.,HIV, syphilis, herpes, gonorrhea, HPV, etc.), blood borne pathogens andpotential bioterrorism agents (e.g., pathogens and toxins in the CDClists of Select A, B and C agents such as B. anthracis, Y. pestis, smallpox, F. tularensis, ricin, botulinum toxins, staph enterotoxins, etc.).Assay panels also include nucleic acid arrays for measuring mRNA levelsof mRNA coding for cytokines, growth factors, components of theapoptosis pathway, expression of the P450 enzymes, expression of tumorrelated genes, pathogens (e.g., the pathogens listed above), etc.Preferred panels also include nucleic acid arrays for genotypingindividuals (e.g., SNP analysis), pathogens, tumor cells, etc. Preferredpanels also include libraries of enzymes and/or enzyme substrates (e.g.,substrates and/or enzymes associated with ubiquitination, proteaseactivity, kinase activity, phosphatase activity, nucleic acid processingactivity, GTPase activity, guanine nucleotide exchange activity, GTPaseactivating activity, etc.). Preferred panels also include libraries ofreceptors or ligands (e.g., panels of G-protein coupled receptors,tyrosine kinase receptors, nuclear hormone receptors, cell adhesionmolecules (integrins, VCAM, CD4, CD8), major bistocompatibility complexproteins, nicotinic receptors, etc.). Preferred panels also includelibraries of cells, cell membranes, membrane fragments, reconstitutedmembranes, organelles, etc. from different sources (e.g., from differentcell types, cell lines, tissues, organisms, activation states, etc.).

In an embodiment, the microfluidic products comprise a component of atest kit. In an embodiment, the microfluidic product comprises and assaycartridge component of a test kit. The test kits may includedisassembled components necessary to make an assay cartridge of theinvention. Alternatively, the kits may comprise, in one or morecontainers, an assay cartridge of the invention and at least oneadditional assay reagent necessary to carry out an assay. The one ormore assay reagents may include, but are not limited to, bindingreagents (preferably, labeled binding reagents, more preferably bindingreagents labeled with electrochemiluminescent labels) specific for ananalyte of interest, ECL coreactants, enzymes, enzyme substrates,extraction reagents, assay calibration standards or controls, washsolutions, diluents, buffers, labels (preferably,electrochemiluminescent labels), etc. In another embodiment, the kitsinclude cartridges of the invention adapted for extracting samples suchas samples collected on applicator sticks. These kits can includeapplicator sticks (more preferably swabs) that have properties that arematched to the specific cartridge. Such kits may also include extractionbuffers for extracting the sample on the applicator stick. The kit mayalso contain (in the cartridge or as a separate component), one or morelabeled binding reagents against markers of pathogens.

In still other embodiments, the present invention provides an apparatuscomprising: a substrate having therein a fluidic channel, the substratecomprising a test area and a reservoir, the reservoir, wherein thefluidic channel fluidically communicating with the test area so that atest compound placed in the reservoir is delivered to the test area. Insome embodiments, the fluidic channel ends in an open port adjacent tothe test area. In further embodiments, the apparatus is configured foruse with an assay system selected from the group consisting ofcolorimetric, liquid crystal, fluorimetric, and densitometric assaysystems. In some embodiments, the reservoir and the fluidic channel areformed from a material selected from the group consisting of glass,polypropylene, polystyrene, and silicone. In other embodiments, theapparatus is configured as an insert for use with a multiwell plate. Infurther embodiments, the apparatus comprises a plurality of test areas,reservoirs, and fluidic channels allowing parallel testing of aplurality of test compounds.

In other embodiments, the microfluidic products of the invention can beused to analyze a predetermined property of a cell or cells comprising:providing a substrate, cells and mesogens; applying the cells to thesurface; and analyzing the cells by contacting the surface and the cellswith the mesogens. Analysis of a variety of predetermined properties iscontemplated, including, but not limited to proliferation in response toa compound, differentiation in response to a compound, and taxis inresponse to compound. In some embodiments, the analyzing step furthercomprises measuring the effect of a substance or compound on the cell.In some embodiments, the effect is quantified.

A detailed description of various physical and chemical assays isprovided in Remington: The Science and Practice of Pharmacy, A. R.Gennaro (ed.), Mack Publishing Company, chap. 29, “Analysis ofMedicinals,” pp. 437-490 (1995) and in references cited therein whilechapter 30 of the same reference provides a detailed description ofvarious biological assays. The assays described include titrimetricassays based on acid-base reactions, precipitation reactions, redoxreactions, and complexation reactions, spectrometric methods,electrochemical methods, chromatographic methods, and other methods suchas gasometric assays, assays involving volumetric measurements andmeasurements of optical rotation, specific gravity, and radioactivity.Other assays described include assays of enzyme-containing substances,proximate assays, alkaloidal drug assays, and biological tests such aspyrogen test, bacterial endotoxin test, depressor substances test, andbiological reactivity tests (in-vivo and in-vitro). Many assays based onfluorescence or changes in fluorescence have been developed and could beperformed using methods and devices of the invention.

In addition, Remington: The Science and Practice of Pharmacy, A. R.Gennaro (ed.), Mack Publishing Company, chap. 31, “Clinical Analysis,”pp. 501-533 (1995) and references cited therein provide a detaileddescription of various methods of characterizations and quantitation ofblood and other body fluids. In particular, the reference includes adetailed description of various tests and assays involving various bodyfluid components such as erythrocytes, hemoglobin, thrombocyte,reticulocytes, blood glucose, nonprotein nitrogen compounds, enzymes,electrolytes, blood-volume and erythropoeitic mechanisms, and bloodcoagulation.

Detection/Analysis Features of Microfluidic Products

Many different techniques exist to identify or measure practically anycharacteristic of a chemical provided that the characteristic orcharacteristics of interest for analysis can be sufficiently identifiedand detected or measured to distinguish chemicals having the desiredcharacteristic(s) from those which do not. In embodiments, themicrofluidic products can comprise an optical detection system. Opticaldetection systems typically include an optical train for directing anoptical signal from the microfluidic channels of the device via theoptical element integrated therein, to an appropriate light detector,such as a photodiode or photomultiplier tube. In some embodiments, thedetector includes a light source for directing an appropriate amount oflight energy at the channels of the device, in order to produce ameasurable optical signal, e.g., fluorescence, absorbance, etc. Examplesof appropriate light sources include, e.g., lasers, laser diodes, LEDs,high intensity lamps, and the like. The detector can be any device ormethod for evaluating a physical characteristic of a fluid as it passesthrough the detection region.

One optical detector can be a microscope, which may be coupled with acomputer and/or other image processing or enhancement devices to processimages or information produced by the microscope using known techniques.For example, molecules can be analyzed and/or sorted by size ormolecular weight. Reactions can be monitored by measuring theconcentration of a product produced or the concentration of a reactantremaining at a given time. Enzymes can be analyzed and/or sorted by theextent to which they catalyze a chemical reaction of an enzyme'ssubstrate (conversely, an enzyme's substrate can be analyzed (e.g.,sorted) based on the level of chemical reactivity catalyzed by anenzyme). Biological particles or molecules such as cells and virions canbe sorted according to whether they contain or produce a particularprotein, by using an optical detector to examine each cell or virion foran optical indication of the presence or amount of that protein. Achemical itself may be detectable, for example by a characteristicfluorescence, or it may be labeled or associated with a tag thatproduces a detectable signal when, for example, a desired protein ispresent, or is present in at least a threshold amount.

To detect a chemical or tag, or to determine whether a chemical or taghas a desired characteristic, the detection region may include anapparatus (e.g., a light source such as a laser, laser diode, highintensity lamp such as mercury lamp) for stimulating a chemical or tagfor that characteristic to, for example, emit measurable light energy.In embodiments where a lamp is used, the channels may be shielded fromlight in all regions except the detection region. In embodiments where alaser is used, the laser can be set to scan across a set of detectionregions. In addition, laser diodes may be fabricated into the samesubstrate that contains the analysis units. Alternatively, laser diodesmay be incorporated into a second substrate (i.e., a laser diode chip)that is placed adjacent to the analysis or sorter substrate such thatthe laser light from the diodes shines on the detection region(s).

Additional Agents that can be Incorporated into Fluid Passages orCoatings Configured to Control Liquid Flow

In embodiments, the surfaces of the fluid passages of the microfluidicproduct can comprise one or more species or agents that provideadditional functions. The coating configured to control liquid flow canalso comprise one or more species or agents that are incorporated intocoating that provide additional functions. These species includetherapeutic agents, detection agents, biomolecules or biopolymers, andcolor agents. The species can comprise polymerized or repeating units ofnucleic acid or amino acid units. Species can be for exampleoligonucleotides, DNA, RNA, protein, peptide, sugar, carbohydrate, andthe like. The species can be a natural species such as for example anatural protein.

In one embodiment, the species can comprise one or more lipids, andlipids are generally known in the art. See for example, Bohinski, ModernConcepts in Biochemistry, 4.sup.th Ed., Chapter 8, “Lipids andBiomembranes.” For example, lipids can be simple lipids, compoundlipids, or derived lipids. Simple lipids can be for exampleacylglycerols or waxes. Compound lipids can be for examplephsphoacylglycerols, sphingomyelins, cerebrosides, or gangliosides.Derived lipids can be for example steroids, carotenoids, or lipidvitamins. Lipids can be used which are natural or synthetic. The lipidcan be able to form liposomes in aqueous solution, either on its own orin combination with other lipids. Lipids can be compounds comprisinglong hydrocarbon chains which can result in them being insoluble inwater but soluble in nonpolar organic solvents. Additional examples oflipids include fats, oils, steroid and waxes.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct can comprise glycerides. In another embodiment, the coatingconfigured to control liquid flow can comprise glycerides. Glyceridesare one type of lipids which are formed from glycerol and fatty acids.Glycerol comprises three hydroxyl groups which upon esterification withone, two or three fatty acids forms monoglycerides, diglycerides andtriglycerides respectively. If one of the fatty acids is replaced with asugar or a phosphate the resulting compound is a glycolipid or aphospholipid respectively. The fatty acids can be unsaturated,saturated, monounsaturated or polyunsaturated. Examples of unsaturatedfatty acids includes, oleic, linoleic, linolenic and arachidonic acid.Examples of saturated fatty acids includes, myristic, palmitic andstearic acids. Further, the fatty acids may adopt a cis or transconfiguration. The length of the fatty acid chain may vary. For example,the fatty acid hydrocarbon chain may comprise more than 3 carbon atoms,between 3-18 atoms or between 12-20 carbon atoms. The chain may or maynot be branched. In one embodiment, the lipid compound comprises aphosphate group. In another embodiment, the lipid compound comprises asugar group. In one embodiment, the lipid compound comprises one, two orthree fatty acids. In a further embodiment, the lipid compound comprisesat least one fatty acid which is saturated, monounsaturated orpolyunsaturated. The lipid can comprise two fatty acids. At least onefatty acid can be monounsaturated. Both fatty acids can bemonounsaturated. The fatty acid may be cis or trans. In one embodiment,at least one fatty acid comprises at least 3 carbon atoms. In anotherembodiment, at least one fatty acid comprises between 3 and 18 carbonatoms, including all integers in between. In another embodiment, atleast one fatty acid comprises between 12 and 20 carbon atoms includingall integers in between.

In embodiments, the lipid can be a phospholipid or a phospholipidderivative. The lipid can exhibit a gel-liquid crystal transitiontemperature. The molecular weight of the lipid can be for example 250 toabout 2,000, or about 500 to about 1,500, or about 500 to about 1,000.Non limiting examples include phophacholine, phosphoglycerol,phosphatidic acid, phosphoserine, PEG phospholipid, and the like. Thelipid can serve as a carrier. In one embodiment, the lipid is1,2-dioleoyl-sn-glycero-3 pphosphocholine (“DOPC”). Other examplesinclude POPC and DM PC. See for example Lenhart et al., Small, 2007, 3,no. 1, 71-75 for lipids which can be patterned.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct can comprise species such as proteinaceous material and proteinsand peptides. In another embodiment, the coating configured to controlliquid flow can comprise species such as proteinaceous material andproteins and peptides. Proteinaceous materials include for exampleantibodies, enzymes, and the like. Types of proteins that can beincorporated include, but are not limited to, enzymes, storage proteins,transport proteins, contractile proteins, protective proteins, toxins,hormones, and structural proteins. Examples of storage proteins include,but are not limited to ovalbumin, casein, ferritin, gliadin, and zein.Examples of transport proteins include, but are not limited tohemoglobin, hemocyanin, myoglobin, serum albumin, .beta.1-lipoprotein,iron-binding globulin, and ceruloplasmin. Examples of contractileproteins include, but are not limited to myosin, actin, dynein. Examplesof protective proteins include, but are not limited to antibodies,complement proteins, fibrinogen, and thrombin. Examples of enzymesinclude, but are not limited to ribonucleases, cytochrome c, lysozymes,proteases, kinases, polymerases, exonucleases, and endonucleases.Enzymes and their binding mechanisms are disclosed, for example, inEnzyme Structure and Mechanism, 2.sup.nd Ed., by Alan Fersht, 1977,including in Chapter 15 the following enzyme types: dehydrogenases,proteases, ribonucleases, staphyloccal nucleases, lysozymes, carbonicanhydrases, and triosephosphate isomerase. Examples of toxins include,but are not limited to, Clostridium botulinum toxin, diptheria toxin,cholera toxin proteins, Alexa Fluor 594 modified cholera toxin proteins,snake venoms, and ricin. Examples of hormones include, but are notlimited to, insulin, adrenocorticotrophic hormone and insulin-likegrowth hormone, and growth hormone. Examples of structural proteinsinclude, but are not limited to, viral-coat proteins, glycoproteins,membrane-structure proteins, .alpha.-keratin, sclerotin, fibroin,collagen, elastin, and mucoproteins. Natural or synthetic peptides andproteins can be used. Proteins that can be used, for example, areprepared by recombinant methods. The protein or peptide can contain asingle polypeptide chain or multiple polypeptide chains. The number ofpeptide bonds in the peptide can be, for example, at least three, ten orless, at least 100, about 100 to about 300, or at least 500. The proteincan be simple or conjugated.

Examples of conjugated proteins include, but are not limited to,nucleoproteins, lipoproteins, phosphoproteins, metalloproteins andglycoproteins. The protein can be a virus, which can be complexes ofproteins and nucleic acids, be they of the DNA or RNA types. The proteincan be a shell to larger structures such as spheres or rod structures.

Proteins can be globular or fibrous in conformation. They can comprise apolypeptide chain or chains arranged in parallel as in, for example, afiber. Examples include collagen and elastin. Globular proteins arepolypeptides that are tightly folded into spherical or globular shapesand are mostly soluble in aqueous systems. Many enzymes, for example,are globular proteins, as are antibodies, some hormones and transportproteins, such as serum albumin and hemoglobin. Proteins can be usedwhich have both fibrous and globular properties, like myosin andfibrinogen, which are tough, rod-like structures but are soluble. Theproteins can possess more than one polypeptide chain, and can beoligomeric proteins, their individual components being called protomers.The oligomeric proteins usually contain an even number of polypeptidechains, not normally covalently linked to one another. Hemoglobin is anexample of an oligomeric protein. Examples of proteins includeimmunoglobulins, IgG (rabbit, human, mouse, and the like), Protein A/G,fibrinogen, fibronectin, lysozymes, streptavidin, avdin, ferritin,lectin (Con. A), and BSA. Rabbit IgG and rabbit anti-IgG, bound insandwich configuration to IgG are useful examples. Spliceosomes andribozomes and the like can be used.

A wide variety of proteins are known to those of skill in the art andcan be used. See, for instance, Chapter 3, “Proteins and theirBiological Functions: A Survey,” at pages 55-66 of BIOCHEMISTRY by A. L.Lehninger, 1970, which is incorporated herein by reference. Otherembodiments include various nucleic acids. For example, the nucleic acidcan be synthetically made, modified to include, for example, functionalgroups tailored for chemisorption or covalent bonding to the substrate,as well as naturally occurring. It can be of low, medium, or highmolecular weight, oligomeric or polymeric. It can be single-, double-,or even triple-stranded. The nucleic acid can be based ondeoxyribonucleic acid (DNA), ribonucleic acid (RNA), or combinationsthereof. The structure of nucleic acids is generally described in, forexample, Calladine and Drew, Understanding DNA, The Molecule and How itWorks, 2.sup.nd Ed., 1997.

General types of nucleic acid that can be incorporated into the surfacesof the fluid passages and/or coatings configured to control liquid flowinclude, for example, DNA, RNA, PNA, CNA, RNA, HNA, p-RNA,oligonucleotides, oligonucleotides of DNA, oligonucleotides of RNA,primers, A-DNA, B-DNA, Z-DNA, polynucleotides of DNA, polynucleotides ofRNA, T-junctions of nucleic acids, domains of non-nucleic acidpolymer-nucleic acid block copolymers, and combinations thereof.Additional general types of nucleic acids include, for example, viralRNA or DNA, a gene associated with a disease, bacterial DNA, fungal DNA,nucleic acid from a biological source, nucleic acid which is a productof a polymerase chain reaction amplification, nucleic acid contactedwith nanoparticles, and nucleic acid double-stranded and hybridized withthe oligonucleotides on the nanoparticles resulting in the production ofa triple-stranded complex.

In general, the nucleic acid can be any of a group of organic substancesfound in cells and viruses that play a central role in the storage andreplication of hereditary information and in the expression of thisinformation through protein synthesis. Purines, pyrimidines,carbohydrates, and phosphoric acid generally characterize thefundamental organic substances of a nucleic acid. Purines andpyrimidines are nucleotides, a nucleoside in which the primary hydroxygroup of either 2-deoxy-D-ribose or of D-ribose is esterified byorthophosphoric acid. A nucleoside is a compound in which a purine orpyrimidine base is bound via a N-atom to C-1 replacing the hydroxy groupof either 2-deoxy-D-ribose or of D-ribose, but without any phosphategroups. The common nucleosides in biological systems are adenosine,guanosine, cytidine, and uridine (which contain ribose) anddeoxyadenosine, deoxyguanosine, deoxycytidine and thymidine (whichcontain deoxyribose). Thus, a purine base may be an adenine nucleotideor a guanine nucleotide. A pyrimidine base may be thymine nucleotide, acytosine nucleotide, or a uracil nucleotide.

The sequence of a nucleic acid may be random or specific so as to encodea desired amino acid structure. For instance, a group of threenucleotides may comprise a codon. One codon comprises an amino acid. Thecoding region of a nucleic acid comprises codons. The nucleic acid canexist freely or can be bound to peptides or proteins to formnucleoproteins in discreet bundles or structured forms such as, forexample, chromosomes. A nucleic acid also can exist in single-strandedor double-stranded forms. A nucleic acid may also be linear, circular,or supercoiled. Nucleic acid may be isolated directly from a cell ororganelle. A plasmid or cloning vector are also examples of nucleicacids.

The nucleic acid can be made up of nucleotides, each containing acarbohydrate sugar (deoxyribose), a phosphate group, and mixtures ofnitrogenous purine- and pyrimidine-bases. The sugar may be of a cyclicor acyclic form. DNA comprises only thymine and cytosine pyrimidines andno uracil. DNA may be isolated from a cell as genomic, nuclear, ormitochondrial DNA, or made synthetically (i.e., by chemical processes).

A gene present in a cell typically comprises genomic DNA made up ofexonic and intronic stretches of DNA. The exonic stretches comprisesnucleotides that comprise codons that encode amino acids, whereas theintronic stretches of DNA comprise nucleotides that likely do notcomprise codons that encode amino acids. The nucleotide sequence ofpurines and pyrimidines determine the sequences of amino acids in thepolypeptide chain of the protein specified by that gene.

DNA may also be isolated as complementary or copy DNA (cDNA) synthesizedfrom an RNA template by the action of RNA-dependent DNA polymerase.

When in double-stranded form, the two DNA strands form a double helix.In this helix, each nucleotide in one strand is hydrogen bonded to aspecific nucleotide on the other strand. Thus, in DNA, adenine bondswith thymine and guanine bonds with cytosine. The ability of nucleotidespresent in each strand to bind to each other determines that the strandswill be complementary, e.g., that for every adenine on one strand therewill be a thymine on the other strand.

RNA can be single-stranded or double-stranded and is transcribed from acell's DNA. An RNA molecule may form a hairpin loop or otherdouble-stranded structures. RNA may be template RNA, messenger RNA(mRNA), total RNA, or transfer RNA (tRNA). polysome. RNA-DNA hybridmolecules can be deposited according to the present invention.Furthermore, protein-nucleic acids, or “peptide nucleic acids” (“PNA”)also may be used. The binding properties exhibited between complementarynucleotides can make nucleic acids useful as probes that can bind toother nucleic acids. Nucleic acids can be labelled and used as probes.By any one of a number of standard labelling techniques, nucleic acidprobes can be used to detect, by hybridization, another nucleic acid.The hybridization can be visualized or detected if the label is, forexample, a fluorescent, radioactive, or enzymatic label. In anembodiment, the coating configured to control liquid flow canincorporate a nucleic acid that is labelled, or modified so as tocomprise a detectable entity, like a fluorescent marker or tag, a goldparticle, streptavidin, digoxigenin, a magnetic bead, or other markersknown to the skilled artisan.

The size of a nucleic acid can range considerably, from the size of afew nucleotides, to an oligonucleotide, or probe, to a polynucleotide,gene, chromosome fragment to entire chromosomes and genomes. Forinstance, a single- or double-stranded nucleic acid may be at least 10-,20-, 30-, 40-, 50-, 60-, 70-, 80-, 90, or 100-nucleotides or base pairs(bp) in length. Larger still, a nucleic acid may be at least 0.2 kb, 0.3kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, or 1.0 kb in size.Indeed, a nucleic acid incorporated into the coating of the presentinvention can be at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8kb, 9 kb, or 10 kb or larger in size. One preferred size range is 1-2kb. The nucleic acid can be a chain of varying length of nucleotides andare typically called polynucleotides or oligonucleotides. Anoligonucleotide is an oligomer generally resulting from linear sequencesof nucleotides. The oligonucleotide can comprise, for example, about 2to about 100, about 2 to about 20, about 10 to about 90, or about 15 toabout 35 nucleotides. In oligonucleotide arrays, about 25-meroligonucleotides can be used. Another particular range is about 60- toabout 80-mers.

Nucleic acid arrays, and the types of nucleic acids used therein, aredescribed for example in A Primer of Genome Science, G. Gibson and S.Muse, 2002, Chapters 3-4 (pages 123-181), which is hereby incorporatedby reference. This reference, for example, describes both cDNAmicroarrays and oligonucleotide arrays, labeling, hybridization, andstatistical analysis. cDNA arrays can be used for monitoring therelative levels of expression of thousands of genes simultaneously.PCR-amplified cDNA fragments (ESTs) can be spotted and probed againstfluorescently or radioactively labeled cDNA. The intensity of the signalobserved can be assumed to be in proportion to the amount of transcriptpresent in the RNA population being studied. Differences in intensityreflect differences in transcript level between treatments. Statisticaland bioinformatic analyses can then be performed, usually with the goalof generating hypotheses that may be tested with established molecularbiological approaches. In other embodiments, the coating configured tocontrol liquid flow can also comprise one or more biomolecules. Suchbiomolecules may include proteins; extracellular matrix proteins such asfibronectin, vitronectin and collagen; soluble proteins such as growthfactors, including vascular endothelial growth factor (VEGF),brain-derived neurotrophic factor (BDNF) or neuronal growth factor(NGF); proteins that are part of a cellular membrane, such assemaphorins, neuropilins, PAR1 receptor, ephrins or plexins; proteinsthat are intracellular; a coupling biomolecule that links a protein orother biomolecule to a substrate such as streptavidin or antibodies;blocking agents to provide space for coupling biomolecules, proteins orother biomolecules to bind to a substrate; antibodies; receptors;ligands; lipids; antigens; full-size proteins; protein domains;peptides; enzymes and/or enzyme substrates; polysaccharides; DNA, RNA,or other nucleic acids; small biomolecules such as nucleotides (e.g.cyclic adenosine monophosphate); fluorescent reporters, small moleculesand drugs, peptides and enzymatic substrates; small molecules that bindcovalently to proteins, peptides or nucleic acids; aggregates ofbiomolecules, small particles or colloids less than 10 micron, less than5 microns, less than 1 micron, less than 500 nm, less than 200 nm, lessthan 100 nm, or less than 50 nm diameter, including quantum dots,superparamagnetic nanoparticles, quantum dots coated with biomoleculesas described above, superparamagnetic nanoparticles coated withbiomolecules as described above, dendrimers coated with biomolecules asdescribed above, glass or silica particles coated with biomolecules asdescribed above, liposomes coated with biomolecules as described above,viruses or phage particles and analogous particles; or any combinationthereof.

Biomolecules include, for example, proteins, peptides, nucleic acids,drugs, lipids, bioactive polymers, bioactive compounds, and anycombination thereof. One or more biomolecules may be covalently ornon-convalently attached to the substrate via a coupling molecule ofvarying length. The one or more of protein biomolecules may includefibronectin, vitronectin, collagen, growth factors, cellular membraneproteins, intracellular proteins, extracellular matrix proteins, solubleproteins, signaling proteins, and any combination thereof. The one ormore of biomolecules may be attached to a particle or colloid includequantum dots, superparamagnetic nanoparticles, dendrimers, glass orsilica particles, liposomes, viruses or phage particles and analogousparticles, and any combination thereof.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct comprise colorants. In embodiments, the colorants areincorporated into the coating that is configured to control fluid flow.In embodiments, the coating configured to control liquid flow can alsocomprise colorants. As used herein, the term “colorant” means anysubstance that imparts color and/or other opacity and/or other visualeffect to the composition. The colorant can be added to the coating inany suitable form. A single colorant or a mixture of two or morecolorants can be used in the coating composition described herein.Example colorants include pigments, dyes and tints, such as those usedin the paint industry and/or listed in the Dry Color ManufacturersAssociation (DCMA), as well as special effect compositions. A colorantcan be organic or inorganic and can be agglomerated or non-agglomerated.In general, the colorant can be present in any amount sufficient toimpart the desired visual and/or color effect.

In embodiments, the colorant can be in the form of a dispersionincluding, but not limited to, a nanoparticle dispersion. Nanoparticledispersions can include one or more highly dispersed nanoparticlecolorants and/or colorant particles that produce a desired visible colorand/or opacity and/or visual effect. Nanoparticle dispersions caninclude colorants such as pigments or dyes having a particle size ofless than 150 nm, such as less than 70 nm, or less than 30 nm.Nanoparticle dispersions can also be produced by crystallization,precipitation, gas phase condensation, and chemical attrition (i.e.,partial dissolution). In order to minimize re-agglomeration ofnanoparticles within the coating, a dispersion of resin-coatednanoparticles can be used. As used herein, a “dispersion of resin-coatednanoparticles” refers to a continuous phase in which discreet “compositemicroparticles”, which comprise a nanoparticle and a resin coating onthe nanoparticle, is dispersed.

Example special effect compositions that may be used include pigmentsand/or compositions that produce one or more appearance effects such asreflectance, pearlescence, metallic sheen, phosphorescence,fluorescence, photochromism, photosensitivity, thermochromism,goniochromism and/or color-change. Additional special effectcompositions can provide other perceptible properties, such as opacityor texture. In a non-limiting embodiment, special effect compositionscan produce a color shift, such that the color of the coating changeswhen the coating is viewed at different angles. In certain non-limitingembodiments, a photosensitive composition and/or photochromiccomposition, which reversibly alters its color when exposed to one ormore light sources, can be used in the coating configured to controlliquid flow. Photochromic and/or photosensitive compositions can beactivated by exposure to radiation of a specified wavelength. When thecomposition becomes excited, the molecular structure is changed and thealtered structure exhibits a new color that is different from theoriginal color of the composition. When the exposure to radiation isremoved, the photochromic and/or photosensitive composition can returnto a state of rest, in which the original color of the compositionreturns. In one non-limiting embodiment, the photochromic and/orphotosensitive composition can be colorless in a non-excited state andexhibit a color in an excited state. Full color-change can appear withinmilliseconds to several minutes, such as from 20 seconds to 60 seconds.Example photochromic and/or photosensitive compositions includephotochromic dyes.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct comprise therapeutic agents. In embodiments, the non-genetictherapeutic agents are incorporated into the coating that is configuredto control fluid flow. In embodiments, therapeutic agents in themicrofluidic products can be used to treat a condition. The amount oftherapeutic agent that is provided in connection with variousembodiments of the present invention can be determined by those ofordinary skill in the art and depends upon the condition to be treated,the nature of the therapeutic agent itself, the avenue by which thedevice is administered to the intended subject, and so forth. In someembodiments, the coating acts as a depot for the therapeutic agent,releasing the therapeutic agent in a controlled manner once themicrofluidic product has been positioned within a patient's body. Inother embodiments, the coating acts as a barrier to control the passageof a therapeutic agent.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct comprise non-generic therapeutic agents. In embodiments, thenon-genetic therapeutic agents are incorporated into the coating that isconfigured to control fluid flow. Exemplary non-genetic therapeuticagents include: (a) anti-thrombotic agents such as heparin, heparinderivatives, urokinase, and PPack (dextrophenylalanine proline argininechloromethylketone); (b) anti-inflammatory agents such as dexamethasone,prednisolone, corticosterone, budesonide, estrogen, sulfasalazine andmesalamine; (c) antineoplastic/antiproliferative/anti-mitotic agentssuch as paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine,epothilones, endostatin, angiostatin, angiopeptin, monoclonal antibodiescapable of blocking smooth muscle cell proliferation, and thymidinekinase inhibitors; (d) anesthetic agents such as lidocaine, bupivacaineand ropivacaine; (e) anti-coagulants such as D-Phe-Pro-Arg chloromethylketone, an RGD peptide-containing compound, heparin, hirudin,antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, prostaglandininhibitors, platelet inhibitors and tick antiplatelet peptides; (f)vascular cell growth promoters such as growth factors, transcriptionalactivators, and translational promotors; (g) vascular cell growthinhibitors such as growth factor inhibitors, growth factor receptorantagonists, transcriptional repressors, translational repressors,replication inhibitors, inhibitory antibodies, antibodies directedagainst growth factors, bifunctional molecules consisting of a growthfactor and a cytotoxin, bifunctional molecules consisting of an antibodyand a cytotoxin; (h) protein kinase and tyrosine kinase inhibitors(e.g., tyrphostins, genistein, quinoxalines); (i) prostacyclin analogs;(j) cholesterol-lowering agents; (k) angiopoietins; (l) antimicrobialagents such as triclosan, cephalosporins, aminoglycosides andnitrofurantoin; (m) cytotoxic agents, cytostatic agents and cellproliferation affectors; (n) vasodilating agents; and (o) agents thatinterfere with endogenous vasoactive mechanisms.

In embodiments, the surfaces of the fluid passages of the microfluidicproduct comprise genetic therapeutic agents. In embodiments, the genetictherapeutic agents are incorporated into the coating that is configuredto control fluid flow. Genetic therapeutic agents include anti-sense DNAand RNA, oligo decoys, as well as DNA coding for: (a) anti-sense RNA,(b) tRNA or rRNA to replace defective or deficient endogenous molecules,(c) angiogenic factors including growth factors such as acidic and basicfibroblast growth factors, vascular endothelial growth factor, epidermalgrowth factor, transforming growth factor, platelet-derived endothelialgrowth factor, platelet-derived growth factor, tumor necrosisfactor.alpha., hepatocyte growth factor and insulin-like growth factor,(d) cell cycle inhibitors including CD inhibitors, and (e) thymidinekinase (“TK”) and other agents useful for interfering with cellproliferation. Cells include cells of human origin (autologous orallogeneic), including stem cells and platelets, or from an animalsource (xenogeneic), which can be genetically engineered if desired todeliver proteins of interest.

Numerous therapeutic agents have been identified as agents that preventrestenosis and other negative occurences. Such agents can beincorporated into the fluid passages and/or coating configured tocontrol liquid flow and include one or more of the following: (a)Ca-channel blockers including benzothiazapines such as diltiazem andclentiazem, dihydropyridines such as nifedipine, amlodipine andnicardapine, and phenylalkylamines such as verapamil, (b) serotoninpathway modulators including: 5-HT antagonists such as ketanserin andnaftidrofuryl, as well as 5-HT uptake inhibitors such as fluoxetine, (c)cyclic nucleotide pathway agents including phosphodiesterase inhibitorssuch as cilostazole and dipyridamole, adenylate/guanylate cyclasestimulants such as forskolin, as well as adenosine analogs, (d)catecholamine modulators including .alpha.-antagonists such as prazosinand bunazosine, beta-antagonists such as propranolol andalpha/beta.-antagonists such as labetalol and carvedilol, (e) endothelinreceptor antagonists, (f) nitric oxide donors/releasing moleculesincluding organic nitrates/nitrites such as nitroglycerin, isosorbidedinitrate and amyl nitrite, inorganic nitroso compounds such as sodiumnitroprusside, sydnonimines such as molsidomine and linsidomine,nonoates such as diazenium diolates and NO adducts of alkanediamines,S-nitroso compounds including low molecular weight compounds (e.g.,S-nitroso derivatives of captopril, glutathione and N-acetylpenicillamine) and high molecular weight compounds (e.g., S-nitrosoderivatives of proteins, peptides, oligosaccharides, polysaccharides,synthetic polymers/oligomers and natural polymers/oligomers), as well asC-nitroso-compounds, O-nitroso-compounds, N-nitroso-compounds andL-arginine, (g) ACE inhibitors such as cilazapril, fosinopril andenalapril, (h) ATII-receptor antagonists such as saralasin and losartin,(i) platelet adhesion inhibitors such as albumin and polyethylene oxide,(j) platelet aggregation inhibitors including aspirin and thienopyridine(ticlopidine, clopidogrel) and GP IIb/IIIa inhibitors such as abciximab,epitifibatide and tirofiban, (k) coagulation pathway modulatorsincluding heparinoids such as heparin, low molecular weight heparin,dextran sulfate and beta-cyclodextrin tetradecasulfate, thrombininhibitors such as hirudin, hirulog,PPACK(D-phe-L-propyl-L-arg-chloromethylketone) and argatroban, FXainhibitors such as antistatin and TAP (tick anticoagulant peptide),Vitamin K inhibitors such as warfarin, as well as activated protein C,(l) cyclooxygenase pathway inhibitors such as aspirin, ibuprofen,flurbiprofen, indomethacin and sulfinpyrazone, (m) natural and syntheticcorticosteroids such as dexamethasone, prednisolone, methprednisoloneand hydrocortisone, (n) lipoxygenase pathway inhibitors such asnordihydroguairetic acid and caffeic acid, (o) leukotriene receptorantagonists, (p) antagonists of E- and P-selectins, (q) inhibitors ofVCAM-1 and ICAM-1 interactions, (r) prostaglandins and analogs thereofincluding prostaglandins such as PGE1 and PGI2 and prostacyclin analogssuch as ciprostene, epoprostenol, carbacyclin, iloprost and beraprost,(s) macrophage activation preventers including bisphosphonates, (t)HMG-CoA reductase inhibitors such as lovastatin, pravastatin,fluvastatin, simvastatin and cerivastatin, (u) fish oils andomega-3-fatty acids, (v) free-radical scavengers/antioxidants such asprobucol, vitamins C and E, ebselen, trans-retinoic acid and SOD mimics,(w) agents affecting various growth factors including FGF pathway agentssuch as bFGF antibodies and chimeric fusion proteins, PDGF receptorantagonists such as trapidil, IGF pathway agents including somatostatinanalogs such as angiopeptin and ocreotide, TGF-beta pathway agents suchas polyanionic agents (heparin, fucoidin), decorin, and TGF-betaantibodies, EGF pathway agents such as EGF antibodies, receptorantagonists and chimeric fusion proteins, TNF-.alpha. pathway agentssuch as thalidomide and analogs thereof, Thromboxane A2 (TXA2) pathwaymodulators such as sulotroban, vapiprost, dazoxiben and ridogrel, aswell as protein tyrosine kinase inhibitors such as tyrphostin, genisteinand quinoxaline derivatives, (x) MMP pathway inhibitors such asmarimastat, ilomastat and metastat, (y) cell motility inhibitors such ascytochalasin B, (z) antiproliferative/antineoplastic agents includingantimetabolites such as purine analogs (e.g., 6-mercaptopurine orcladribine, which is a chlorinated purine nucleoside analog), pyrimidineanalogs (e.g., cytarabine and 5-fluorouracil) and methotrexate, nitrogenmustards, alkyl sulfonates, ethylenimines, antibiotics (e.g.,daunorubicin, doxorubicin), nitrosoureas, cisplatin, agents affectingmicrotubule dynamics (e.g., vinblastine, vincristine, colchicine,paclitaxel and epothilone), caspase activators, proteasome inhibitors,angiogenesis inhibitors (e.g., endostatin, angiostatin and squalamine),rapamycin, cerivastatin, flavopiridol and suramin, (aa) matrixdeposition/organization pathway inhibitors such as halofuginone or otherquinazolinone derivatives and tranilast, (bb) endothelializationfacilitators such as VEGF and RGD peptide, (cc) blood rheologymodulators such as pentoxifylline, and (dd) endothelial-cell specificmitogens.

Typical polynucleotide therapeutic agents generally include DNA encodingfor various polypeptide and protein products including those previouslylisted. Some additional examples of polynucleotide therapeutic agentsinclude DNA encoding for the following: cytokines such as colonystimulating factors (e.g., granulocyte-macrophage colony-stimulatingfactor), tumor necrosis factors (e.g., fas ligand) and interleukins(e.g., IL-10, an anti-inflammatory interleukin), as well as proteaseinhibitors, particularly serine protease inhibitors (e.g., SERP-1),tissue inhibiting metalloproteinases (e.g., TIMP-1, TIMP-2, TIMP-3,TIMP-4), monocyte chemoattractant proteins (e.g., MCP-1), protein kinaseinhibitors including cyclin-dependent kinase inhibitors (e.g., p27,p21), endogenous and inducible nitric oxide synthase, CO-generatingenzymes, such as hemoxygenases, which catalyze the oxidation of hemeinto the biologically active molecules iron biliverdin and CO (e.g.,HOI-1), antiproliferative compounds, such as hKIS in a transdominantmutant peptide form, which are capable of interfering with the abilityof endogenous hKIS to phosphorylate p27 thereby enhancing cell cyclearrest, as well as derivatives

Additional embodiments of the invention include the following:

Embodiment 1: A microfluidic product comprising one or more fluidpassages wherein a first fluid passage comprises a top and a bottomsurface wherein the first fluid passage comprises a coating configuredto control liquid flow wherein the coating configured to control liquidflow comprises a gradient surface energy coating from a proximallocation to a distal location on a surface of the fluid passage.

Embodiment 2: A microfluidic product comprising a plurality of fluidpassages wherein the plurality of fluid passages comprise a first fluidpassage and a second fluid passage, each with a top and a bottomsurface, wherein both the first fluid passage and the second fluidpassage each comprise a coating configured to control liquid flowwherein the coating configured to control liquid flow comprises agradient surface energy coating from a proximal location to a distallocation on a surface of the fluid passage.

Embodiment 3: A microfluidic product of embodiments 1 and 2 wherein atleast one fluid passage comprises a coating configured to control liquidflow wherein the difference between the contact angle formed with waterand the surface at the proximal location of the gradient surface energycoating and the contact angle formed with water and the surface at thedistal location of the gradient surface energy coating is no less than10 degrees.

Embodiment 4: A microfluidic product of embodiments 1-3 furthercomprising a fluid passage that is not coated.

Embodiment 5: A microfluidic product of embodiments 1-4, wherein thelinear velocity of liquid in a first fluid passage is no less than 10percent higher than the linear velocity of liquid in a second fluidpassage.

Embodiment 6: A microfluidic product of embodiments 1-5 wherein at leasta first fluid passage and the second fluid passage are in fluidcommunication with each other.

Embodiment 7: A microfluidic product of embodiments 1-6 wherein thechemical composition of the coating in a first fluid passage isdifferent from the chemical composition of the coating in a fluidpassage.

Embodiment 8: A microfluidic product of embodiments 1-7 wherein two ormore fluid passages comprise a coating configured to control liquid flowwherein the coating comprises a gradient surface energy coating from aproximal location to a distal location on a surface of the fluid passagewherein the chemical composition of the coating is the same for the twoor more fluid passages.

Embodiment 9: The microfluidic product of embodiments 1-8 furthercomprising at least one inlet port in communication with a first channeland a detection region in fluid communication of the first channel.

Embodiment 10: The microfluidic product of embodiment 9 furthercomprising a detector associated with the detection region.

Embodiment 11: The microfluidic product of embodiments 1-10 wherein thelength of a first channel is no more than 10 percent greater than thelength of a second channel.

Embodiment 12: The microfluidic product of embodiments 1-11 wherein thewidth of a first channel is no more than 10 percent greater than thewidth of a second channel.

Embodiment 13: The microfluidic product of embodiments 1-12 wherein theheight of a first channel is no more than 10 percent greater than theheight of a second channel.

Embodiment 14: A microfluidic product of embodiments 1-13 wherein thefluid passages comprise rectangular channels.

Embodiment 15: A microfluidic product of embodiment 14 wherein thelength of the channels is no less than 3 millimeters, and the width ofthe channels is no greater than 1 millimeters.

Embodiment 16: The microfluidic product of embodiments 1-15 wherein thecoating configured to control liquid flow comprises a species having afunctional group M1 and a species having a functional group M2 where M1and M2 have different surface energies.

Embodiment 17: The microfluidic product of embodiment 16 wherein thecoating comprises a monolayer coating.

Embodiment 18: The microfluidic product of embodiments 1-17, wherein thecoating is formed from species X1-J1-M1 and X2-J2-M2 wherein X1, X2, M1,and M2 represent separate functional groups where M1 and M2 havedifferent surface energies and J1 and J2 represents spacer moieties, thespecies X1-J1-M1 and X2-J2-M2 forming a coating onto the surface fromsolution.

Embodiment 19: The microfluidic product of embodiments 1-18 wherein themolar concentration of the species comprising the functional group M2continuously increases relative to the concentration of the speciescomprising functional group M1 in the coating from the proximal locationto the distal location.

Embodiment 20: The microfluidic product of embodiments 1-19 wherein thecoating configured to control liquid flow comprises a photoreactivegroup.

Embodiment 21: The microfluidic product of embodiments 1-20 wherein thecoating configured to control liquid flow comprises a thermoreactivegroup.

Embodiment 22: The microfluidic product of embodiments 1-21 wherein thecontact angle formed with water and the surface at the proximal locationof the gradient surface energy coating is between 90 and 120 degrees andthe contact angle formed with water and the surface at the distallocation of the gradient surface energy coating is between 10 and 110degrees.

Embodiment 23: The microfluidic product of embodiments 1-22 wherein thecontact angle formed with water and the surface at the proximal locationof the gradient surface energy coating is between 90 and 120 degrees andthe contact angle formed with water and the surface at the distallocation of the gradient surface energy coating is between 10 and 70degrees.

Embodiment 24: The microfluidic product of embodiments 1-23 wherein thecontact angle formed with water and the surface at the proximal locationof the gradient surface energy coating is between 90 and 120 degrees andthe contact angle formed with water and the surface at the distallocation of the gradient surface energy coating is between 10 and 40degrees.

Embodiment 25: The microfluidic product of embodiments 1-24 wherein thecontact angle formed with water and the surface at the proximal locationof the gradient surface energy coating is between 60 and 120 degreesEmbodiment 26: The microfluidic product of embodiments 1-25 wherein thecontact angle formed with water and the surface at the proximal locationof the gradient surface energy coating is between 40 and 120 degrees.

Embodiment 27: The microfluidic product of embodiments 1-26 wherein thecontact angle formed with water and the surface at the distal locationof the gradient surface energy coating is between 10 and 40 degrees.

Embodiment 28: The microfluidic product of embodiments 1-27 wherein thecontact angle formed with water and the surface at the distal locationof the gradient surface energy coating is between 10 and 70 degrees.

Embodiment 29: The microfluidic product of embodiments 1-28 wherein thecontact angle formed with water and the surface at the distal locationof the gradient surface energy coating is between 10 and 110 degrees.

Embodiment 30: The microfluidic product of embodiments 1-29 wherein theproximal location corresponds to the fluid passage entrance and thedistal location corresponds to the fluid passage exit.

Embodiment 31: The microfluidic product of embodiments 1-30 wherein theproximal location corresponds to a location on a surface of the fluidpassage that is downstream of the fluid passage entrance.

Embodiment 32: The microfluidic product of embodiments 1-31 wherein theproximal location corresponds to a location on the surface of the fluidpassage that is at the mid-point between the fluid passage entrance andthe fluid passage exit

Embodiment 33: The microfluidic product of embodiments 1-31 wherein theproximal location corresponds to a location on the surface of the fluidpassage that is upstream of the mid-point between the fluid passageentrance and the fluid passage exit.

Embodiment 34: The microfluidic product of embodiments 1-31 wherein theproximal location corresponds to a location on the surface of the fluidpassage that is downstream of the mid-point between the fluid passageentrance and the fluid passage exit.

Embodiment 35: The microfluidic product of embodiments 1-34 wherein thedistal location corresponds to a location on the surface of the fluidpassage upstream of the fluid passage exit.

Embodiment 36: The microfluidic product of embodiments 1-33 wherein thedistal location corresponds to a location on the surface of the fluidpassage that is at the mid-point between the fluid passage entrance andthe fluid passage exit.

Embodiment 36: The microfluidic product of embodiments 1-33 and 35wherein the distal location corresponds to a location on the surface ofthe fluid passage that is upstream of the mid-point between the fluidpassage entrance and the fluid passage exit.

Embodiment 37: The microfluidic product of embodiments 1-35 wherein thedistal location corresponds to a location on the surface of the fluidpassage that is downstream of the mid-point between the fluid passageentrance and the fluid passage exit.

Embodiment 38: The microfluidic product of embodiments 1-37 wherein asurface of the fluid passages comprise a polymer.

Embodiment 39: The microfluidic product of embodiments 1-38 wherein themicrofluidic product comprises a thermoplastic polymer.

Embodiment 40: The microfluidic product of embodiments 1-39 wherein asurface of the fluid passages comprise a metal selected from the groupconsisting of gold, silver, nickel, copper, steel, palladium, platinum,or their alloys.

Embodiment 41: The microfluidic product of embodiments 1-40 wherein asurface of the fluid passages comprise a glass, metal oxide, ordielectric material.

Embodiment 42: The microfluidic product of embodiments 1-41, wherein asurface of a fluid passage further comprises a substantially uniformcoating.

Embodiment 43: The microfluidic product of embodiments 42 wherein thesubstantially uniform coating is a hydrophilic coating.

Embodiment 44: The microfluidic product of embodiments 42-43 wherein thesubstantially uniform coating is a hydrophobic coating.

Embodiment 45: The microfluidic product of embodiments 1-44 wherein forat least one fluid passage, the contact angle formed with water and thesurface of the fluid passage at the proximal location of the gradientsurface energy coating is no less than 90 degrees and wherein thecontact angle formed with water and the surface decreases at an averagelinear rate for the length of the fluid passage.

Embodiment 46: The microfluidic product of embodiments 1-45 wherein forat least one fluid passage, the contact angle formed with water and thesurface of the fluid passage at the proximal location of the gradientsurface energy coating is no less than 60 degrees and wherein thecontact angle formed with water and the surface decreases at an averagelinear rate for the length of the fluid passage.

Embodiment 47: The microfluidic product of embodiments 1-46 wherein forat least one fluid passage, the contact angle formed with water and thesurface of the fluid passage at the proximal location of the gradientsurface energy coating is no less than 30 degrees and wherein thecontact angle formed with water and the surface decreases at an averagelinear rate for the length of the fluid passage.

Embodiment 48: The microfluidic product of embodiments 45-47 wherein theaverage linear rate is between 1 degree/millimeter and 10degrees/millimeter.

Embodiment 49: The microfluidic product of embodiments 1-48 wherein themicrofluidic product comprises a clear or transparent material.

Embodiment 50: The microfluidic product of embodiments 1-49 furtherwherein the material comprising the fluid passages comprises a scale formeasuring length.

Embodiment 51: The microfluidic product of embodiments 1-50 wherein theoverall length of the device is no more than 200 millimeters, theoverall width is no more than 150 millimeters, and the overall thicknessis no more than 20 millimeters.

Embodiment 52: The microfluidic product of embodiments 1-51 wherein theoverall length of the device is no more than 100 millimeters, theoverall width is no more than 75 millimeters, and the overall thicknessis no more than 10 millimeters.

Embodiment 53: The microfluidic product of embodiments 1-52 wherein theheight of the fluid passages is no greater than 200 microns.

Embodiment 54: The microfluidic product of embodiments 42-53 wherein theuniform coating is between the fluid passage entrance and the proximallocation for the gradient surface energy coating.

Embodiment 55: The microfluidic product of embodiments 42-54 wherein theuniform coating is between the fluid passage exit and the proximallocation for the gradient surface energy coating.

Embodiment 56: The microfluidic product of embodiments 42-55 wherein theuniform coating is between the fluid passage exit and the distallocation for the gradient surface energy coating.

Embodiment 57: The microfluidic product of embodiments 1-56 wherein themicrofluidic product comprises an assay cartridge.

Embodiment 58: The microfluidic product of embodiments 1-57 wherein themicrofluidic product comprises a sample module.

Embodiment 59: The microfluidic product of embodiments 1-58 wherein thefluid microfluidic products comprises a reagent module.

Embodiment 60: The microfluidic product of embodiments 1-59 wherein afluid passage comprises a therapeutic agent.

Embodiment 61: The microfluidic product of embodiments 1-60 wherein afluid passage comprises a detection agent.

Embodiment 62: The microfluidic product of embodiments 1-61 wherein afluid passage comprises a colorant.

Embodiment 63: The microfluidic product of embodiments 1-62 wherein afluid passage comprises a biopolymer.

Embodiment 64: The microfluidic product of embodiments 1-63 wherein thecoating configured to control liquid flow comprises a therapeutic agent.

Embodiment 65: The microfluidic product of embodiments 1-64 wherein thecoating configured to control liquid flow comprises a detection agent.

Embodiment 66: The microfluidic product of embodiments 1-65 wherein thecoating configured to control liquid flow comprises a colorant.

Embodiment 67: The microfluidic product of embodiments 1-66 wherein thecoating configured to control liquid flow comprises a biopolymer.

The above disclosure is intended to be illustrative and not exhaustive.This description will suggest many variations and alternatives to one ofordinary skill in this art. All these alternatives and variations areintended to be included within the scope of the claims where the term“comprising” means “including, but not limited to”. Those familiar withthe art may recognize other equivalents to the specific embodimentsdescribed herein which equivalents are also intended to be encompassedby the claims.

Further, the particular features presented in the dependent claims canbe combined with each other in other manners within the scope of theinvention such that the invention should be recognized as alsospecifically directed to other embodiments having any other possiblecombination of the features of the dependent claims. For instance, forpurposes of claim publication, any dependent claim which follows shouldbe taken as alternatively written in a multiple dependent form from allprior claims which possess all antecedents referenced in such dependentclaim if such multiple dependent format is an accepted format within thejurisdiction (e.g. each claim depending directly from claim 1 should bealternatively taken as depending from all previous claims). Injurisdictions where multiple dependent claim formats are restricted, thefollowing dependent claims should each be also taken as alternativelywritten in each singly dependent claim format which creates a dependencyfrom a prior antecedent-possessing claim other than the specific claimlisted in such dependent claim below.

Those skilled in the art may recognize other equivalents to the specificembodiment described herein which equivalents are intended to beencompassed by the claims attached hereto.

The following documents contain additional information on the materials,processes, and product applications that can be suitable with themicrofluidic products of the invention. All documents cited in thisapplication are herein incorporated by reference in their entirety.

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We claim: 1) A microfluidic product comprising an inlet port, a firstdetection region positioned downstream of a first fluid passage, and asecond detection region positioned downstream of a second fluid passage,the first and second fluid passages each having a top surface and abottom surface, wherein the first and second fluid passages eachcomprise a coating configured to control liquid flow on the top surfaceor the bottom surface wherein the coating configured to control liquidflow comprises a gradient surface energy coating from a proximallocation on the top or bottom surface to a distal location on the top orbottom surface of the first and second fluid passages, wherein the inletport is in fluid communication with the first and second detectionregions, and the gradient surface energy coating of the first and secondfluid passages comprises species X1-J1-M1 and species X2-J2-M2 whereinX1, X2, M1, and M2 represent separate functional groups where M1 and M2have different surface energies and J1 and J2 represents spacermoieties, and the molar concentration of the species X2-J2-M2 increasesrelative to the molar concentration of the species X1-J1-M1 in thegradient surface energy coating from the proximal location to the distallocation. 2) The microfluidic product of claim 1 wherein the first andsecond detection regions comprises one or more binding domains havingimmobilized binding reagents. 3) The microfluidic product of claim 2wherein the binding reagents comprise one or antibodies, proteins,receptors, ligands, haptens, or nucleic acids. 4) The microfluidicproduct of claim 2 wherein the binding reagents comprise one or moreelectrochemiluminescent labels. 5) The microfluidic product of claim 1further comprising a pierceable container. 6) The microfluidic productof claim 1 further comprising a reagent chamber for storing liquidreagents, the reagent chamber connected via one or more additional fluidpassages to the first or second detection region. 7) The microfluidicproduct of claim 1 wherein the coating configured to control liquid flowfurther comprises a therapeutic agent, a detection agent, a colorant, abiopolymer or combinations thereof. 8) The microfluidic product of claim1 wherein the composition of the gradient surface energy coating in thefirst fluid passage is different from the composition of the gradientsurface energy coating in the second fluid passage. 9) The microfluidicproduct of claim 1 wherein the top or bottom surface of the first orsecond fluid passage comprises a polymer material selected from thegroup consisting of polystyrene, polycarbonate, polyester, polyethylene,polyethylene terephthalate (PET), polyglycolic acid (PGA), polyolefin,poly-(pphenyleneterephthalamide), polyphosphazene, polypropylene,polytetrafluoroethylene, polyurethane, polyvinyl chloride, andpolyacrylate (including polymethacrylate) and the functional groups X1and X2 in species X1-J1-M1 and X2-J2-M2 comprise a photoreactive group.10) The microfluidic product of claim 1 wherein the top surface orbottom surface of the first or second fluid passage comprises a materialselected from the group consisting of gold, silver, nickel, copper,aluminum, cadmium, zinc, palladium, platinum, mercury, lead, iron,chromium, manganese, tungsten, silicon, silicon oxide, and any alloys ofthe above and the functional groups X1 and X2 in species X1-J1-M1 andX2-J2-M2 are selected from the group consisting of thiols, sulfides,disulfides, silanes, and chlorosilanes. 11) The microfluidic product ofclaim 1 wherein the first or second fluid passage further comprises atherapeutic agent, a detection agent, a colorant, a biopolymer orcombinations thereof. 12) The microfluidic product of claim 1 whereinthe M2 species in the surface energy gradient coating of the first fluidpassage is different from the M2 species in the surface energy gradientcoating of the second fluid passage. 13) The microfluidic product ofclaim 2 wherein the binding reagents comprise virus antibodies. 14) Themicrofluidic product of claim 1 wherein the proximal locationcorresponds to the first or second fluid passage entrance and the distallocation corresponds to the first or second fluid passage exit. 15) Themicrofluidic product of claim 1 wherein the proximal locationcorresponds to a location on a surface of the first or second fluidpassage that is downstream of the first or second fluid passageentrance. 16) The microfluidic product of claim 1 wherein the distallocation corresponds to a location on the surface of the first or secondfluid passage upstream of the first or second fluid passage exit. 17)The microfluidic product of claim 1 further comprising a fluid passagethat is not coated. 18) The microfluidic product of claim 1 furthercomprising a third detection region wherein the third detection regionis used in a control/calibration for measuring assay control/calibrationsamples. 19) The microfluidic product of claim 1 wherein themicrofluidic product comprises an assay cartridge used to carry outpanels of assays. 20) The microfluidic product of claim 19 wherein theassay comprises virus antibodies.